Alpha-amino pateamine A derivatives and methods for treating chronic lymphocytic leukemia

ABSTRACT

Pateamine A derivatives and pharmaceutical compositions that include the derivatives. The pateamine A derivatives are α-amino pateamine A derivatives that lack the C5-methyl group of pateamine A.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/563,891, filed Oct. 2, 2017, now U.S. Pat. No. 10,407,444, which isthe national stage of International Application No. PCT/US2016/025355,filed Mar. 31, 2016, which claims the benefit of Application No.62/140,987, filed Mar. 31, 2015. Each application is expresslyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to α-amino pateamine A derivatives andmethods for treating chronic lymphocytic leukemia using the α-aminopateamine A derivatives.

BACKGROUND OF THE INVENTION

Effective new cancer therapies have been developed based on agents withnovel mechanisms of action that specifically target the pathophysiologyof malignances. Most of these diseases exhibit a pronounced defect innormal lymphocyte cell death mechanisms due to over-expression ofpro-survival proteins. It is now recognized that a critical aspect of Bcell malignancy metabolism is directed at replenishing the pro-survivalproteins that keep these cells from dying due to apoptosis. This isrequired because sequence motifs intrinsic to the primary proteinstructure of these pro-survival proteins, signal for the rapid turnoverof these proteins (e.g. Mcl-1, XIAP). This is a hallmark of thepathophysiology of B cell malignancies. Importantly, even transientinhibition of translation rapidly diminishes these key proteins to alevel that cannot prevent apoptosis. Once initiated, this lethal processis irreversible. Because normal lymphoid cells do not exhibit thisdependency, it appears that CLL cells are “addicted” to the continualexpression of the anti-apoptotic proteins for survival.

Pateamine A (PatA) was initially isolated from the marine sponge Mycalesp. by bioassay-guided fractionation based on its cytotoxic activityagainst P388 murine leukemia cells (IC₅₀, 0.27 nmol/L). Consistent withits cytotoxicity, PatA was subsequently shown to induce apoptosis inseveral cancer cell lines. Des-methyl, des-amino pateamine A (DMDAPatA)is a simplified analog of the natural product that is easier tosynthesize and a potent anti-proliferative agent in vitro against >30human cancer cell lines.

PatA and DMDAPatA inhibit cap-dependent translation initiation bysequestration of eIF4A that prevents formation of the eIF4F complex, orby stalling the initiation complex on mRNA. Xenograft studies in miceshowed DMDAPatA has high activity in models of human leukemia andmelanoma leading to significant tumor reduction, thus demonstrating goodbioavailability. The synthesis of >20 derivatives of PatA led to theidentification of DMDAPatA which was also found to be significantly morestable than the natural product. Overexpression of multidrug resistantprotein did not affect this activity. Importantly, DMDAPatA reduces thelevels of intrinsically short-lived anti-apoptotic proteins in primaryCLL cells, and initiates apoptosis. However, preliminary data onDMDAPatA suggests that it is highly protein bound in human plasma andmay lack sufficient in vivo potency required for development as aneffective therapeutic agent.

Although PatA and DMDAPatA appear to be attractive candidates for thedevelopment of therapeutic agents, a need exists for improved PatAderivatives having therapeutic effectiveness, low toxicity, andadvantageous pharmacokinetic properties.

The present invention seeks to fulfill these needs and provides furtherrelated advantages.

SUMMARY OF THE INVENTION

The present invention provides α-amino pateamine A derivatives,pharmaceutical compositions that include the derivatives, an methods forusing the derivatives.

In one aspect, the invention provides α-amino pateamine A derivatives.

In one embodiment, the invention provides pateamine A derivatives havingformula (I), stereoisomers, racemates, and pharmaceutically saltsthereof:

wherein

X is selected from O, NH, and S; and

Y is selected from R, OR, SR, and N(R¹)R²,

wherein R is selected from C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl,and C3-C12 alkyl groups in which one or more carbons are replaced with Oor N atoms, and

wherein R¹ and R² are independently selected from hydrogen, C1-C6 alkyl,C1-C6 haloalkyl, C6-C10 aryl, and C3-C12 alkyl groups in which one ormore carbons are replaced with O or N atoms.

In certain embodiments, the invention provides pateamine A derivativeshaving formula (IIA) and pharmaceutically salts thereof:

wherein X, Y, R, R¹, and R² are as above for formula (I).

In other embodiments, the invention provides pateamine A derivativeshaving formula (IIB) and pharmaceutically salts thereof:

wherein X, Y, R, R¹, and R² are as above for formula (I).

Representative salts of the invention have formula (III):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion.

In certain embodiments, representative salts of the invention haveformula (IVA):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion.

In other embodiments, representative salts of the invention have formula(IVB):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion.

In another embodiment, the invention provides pateamine A derivativeshaving formula (V), stereoisomers, racemates, and pharmaceutically saltsthereof:

wherein

Z is selected from R and OR¹, wherein R and R¹ are as described abovefor formulae (I)-(IV).

In certain embodiments, the invention provides pateamine A derivativeshaving formula (VIA) and pharmaceutically salts thereof:

wherein Z is as described above for formula (V).

In other embodiments, the invention provides pateamine A derivativeshaving formula (VIB) and pharmaceutically salts thereof:

wherein Z is as described above for formula (V).

Representative salts of the invention have formula (VII):

wherein Z is as described above for formulae (V) and A⁻ is apharmaceutically acceptable counter ion.

In certain embodiments, representative salts of the invention haveformula (VIIIA):

wherein Z is as described above for formula (V) and A⁻ is as describedabove for formula (VII).

In other embodiments, representative salts of the invention have formula(VIIIB):

wherein Z is as described above for formula (V) and A⁻ is as describedabove for formula (VII).

In another aspect, the invention provides antibody drug conjugates forthe delivery of the α-amino pateamine derivatives of the invention.

In a further aspect, the invention provides pharmaceutical compositionsthat include a compound of the invention (i.e., a compounds of formulae(I), (II), (III), (IV), (V), (VI), (VII), or (VIII)) and apharmaceutically acceptable carrier. The invention also providespharmaceutical compositions that include an antibody conjugate of theinvention (i.e., an antibody conjugate that delivers a compound offormulae (I), (II), (III), (IV), (V), (VI), (VII), or (VIII)) and apharmaceutically acceptable carrier.

In another aspect, the invention provides a method for inhibiting growthof chronic lymphocytic leukemia (CLL) cells. In the method, growth ofCLL cells is inhibited by contacting CLL cells with an α-amino pateaminecompound of the invention. In certain embodiments, the method iseffective for inhibiting growth of chronic lymphocytic leukemia (CLL)cells in a subject (e.g., a human subject).

In a further aspect, the invention provides a method for treatingchronic lymphocytic leukemia (CLL). In the method, CLL is treated byadministering an effective amount of an α-amino pateamine compound ofthe invention to a subject (e.g., a human subject) in need thereof.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings.

FIGS. 1A-1D are schematic illustrations showing the preparation of arepresentative α-amino pateamine A derivative of the invention (MZ579).

FIGS. 2A-2D compare IC₅₀ dose response curves (% survival of chroniclymphocytic leukemia (CLL) cells as a function of agent concentration(μM)) for pateamine A (PatA) (2A), desmethyl desamino pateamine A(DMDAPatA) (2B), α-amino pateamine A derivative (MZ578) (2C), andα-amino pateamine A derivative (MZ579) (2D) in 10% fetal bovine serum(FBS), 10% human plasma, and 50% human plasma.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides α-amino pateamine A derivatives,pharmaceutical compositions that include the derivatives, an methods forusing the derivatives.

The compounds of the invention are simplified analogs of pateamine Athat lack the C5-methyl group. The compounds of the invention areα-amino (2-amino) pateamine A derivatives.

The compounds of the invention are derivatives of the translationinhibitor pateamine A and as pateamine A derivatives, the compounds ofthe invention are expected to provide anticancer and antiproliferativeeffects by inhibition of eIF4A-dependent translation initiation.

The compounds of the invention display potent inhibitory activityagainst chronic lymphocytic leukemia (CLL) cells and lower plasmaprotein binding (PPB) in human plasma. The combination of potency andlow PPB render the compounds candidates for development of therapeuticagents for treatment of CLL.

The inhibition of translation in CLL cells has the potential forclinical development of therapies for B cell malignancies and toovercome drug resistance to existing standard of care therapeutics.Relapsed refractory CLL remains a clinical problem associated with pooroverall survival. Due to their unique mode of action, inhibition oftranslation and protein biosynthesis, the compounds of the invention maybe useful for combating resistant forms of CLL.

α-Amino Pateamine Derivatives

In one embodiment, the invention provides pateamine A derivatives havingformula (I), stereoisomers, racemates, and pharmaceutically saltsthereof:

wherein

X is selected from O, NH, and S; and

Y is selected from R, OR¹, SR¹, and N(R¹)R²,

wherein R is selected from C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl,and C3-C12 alkyl groups in which one or more carbons are replaced with Oor N atoms, and

wherein R¹ and R² are independently selected from hydrogen, C1-C6 alkyl,C1-C6 haloalkyl, C6-C10 aryl, and C3-C12 alkyl groups in which one ormore carbons are replaced with O or N atoms.

In certain embodiments, the invention provides pateamine A derivativeshaving formula (IIA) and pharmaceutically salts thereof:

wherein X, Y, R, R¹, and R² are as above for formula (I).

In other embodiments, the invention provides pateamine A derivativeshaving formula (IIB) and pharmaceutically salts thereof:

wherein X, Y, R, R¹, and R² are as above for formula (I).

Pharmaceutically acceptable salts may be formed from compounds offormulae (I) and (II) and a pharmaceutically acceptable organic acids(e.g., carboxylic acids) or inorganic acid (e.g., mineral acids).Representative acids include hydrochloric acid, sulfuric acid,phosphoric acid, formic acid, acetic acid, trifluoroacetic acid, maleicacid, fumaric acid, succinic acid, tartaric acid, oxalic acid, citricacid, malic acid, benzoic acid, toluenesulfonic acid, methanesulfonicacid, and benzenesulfonic acid. Such salts may be formed during or afterthe synthesis of the compounds of formulae (I) or (II).

Representative salts of the invention have formula (III):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion. Suitable counter ionsinclude chloride, bromide, iodide, sulfate, phosphate, formate, acetate,trifluoroacetate, maleate, fumarate, succinate, tartrate, oxalate,citrate, malate, benzoate, toluenesulfonate, methanesulfonate, andbenzenesulfonate.

In certain embodiments, representative salts of the invention haveformula (IVA):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion.

In other embodiments, representative salts of the invention have formula(IVB):

wherein X and Y are as described above for formulae (I) and (II), and A⁻is a pharmaceutically acceptable counter ion.

Suitable counter ions include chloride, bromide, iodide, sulfate,phosphate, formate, acetate, trifluoroacetate, maleate, fumarate,succinate, tartrate, oxalate, citrate, malate, benzoate,toluenesulfonate, methanesulfonate, and benzenesulfonate.

For compounds of formulae (I), (II), (III), and (IV), C1-C6 alkyl groupsinclude straight chain (i.e., methyl, ethyl, n-propyl, n-butyl,n-pentyl, and n-hexyl), branched (e.g., s-propyl, s-butyl, t-butyl,s-pentyl, and s-hexyl), and cycloalkyl (i.e., cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl) group;

C1-C6 haloalkyl groups include C1-C6 alkyl groups further substitutedwith one or more halo (e.g., fluoro or chloro) groups (e.g.,trifluoromethyl or trichloromethyl);

C6-C10 aryl groups include phenyl groups optionally substituted with oneof more alkyl groups (e.g., methyl, ethyl);

C3-C12 alkyl groups refer to C3-C12 alkyl groups in which one or morecarbons are replaced with O or N atoms include ether-containing groupsand amine-containing groups that may impart increased water solubilityto the compounds. Representative ether-containing groups include—CH₂—O—CH₃, —CH₂CH₂—O—CH₃, and —CH₂CH₂—O—CH₂CH₂—O—CH₃. Representativeamine-containing groups include —CH₂—NH—CH₃, —CH₂—N(CH₃)₂,—CH₂CH₂—NH—CH₃, and —CH₂CH₂—N(CH₃)₂.

The invention provides compounds of formulae (I), (IIA), (IIB), (III),(IVA) and (IVB) including the following embodiments:

X is O and Y is R (amides), in certain of these embodiments, R ismethyl, trifluoromethyl, or t-butyl;

X is O and Y is OR¹ (carbamates), in certain of these embodiments, R¹ ismethyl or t-butyl;

X is O and Y is N(R¹)R² (ureas), in certain of these embodiments, R¹ ishydrogen and R² is hydrogen, or R¹ is hydrogen and R² is methyl;

X is O and Y is SR¹, in certain of these embodiments, R¹ is methyl ort-butyl;

X is S and Y is R (thioamides), in certain of these embodiments, R ismethyl;

X is S and Y is OR¹ (thiocarbamates), in certain of these embodiments,R¹ is methyl or t-butyl;

X is S and Y is N(R¹)R² (thioureas), in certain of these embodiments, R¹is hydrogen and R² is hydrogen, or R¹ is hydrogen and R² is methyl;

X is S and Y is SR¹, in certain of these embodiments, R¹ is methyl ort-butyl;

X is NH and Y is R, in certain of these embodiments, R is methyl,trifluoromethyl, or t-butyl;

X is NH and Y is OR¹, in certain of these embodiments, R¹ is methyl ort-butyl;

X is NH and Y is N(R¹)R², in certain of these embodiments, R¹ ishydrogen and R² is hydrogen, or R¹ is hydrogen and R² is methyl; and

X is NH and Y is SR¹, in certain of these embodiments, R¹ is methyl ort-butyl.

The preparation of a representative α-amino pateamine A amide derivativeof the invention is described in Example 1 and illustrated in FIG. 1(MZ579).

The preparation of another representative pateamine A amide derivativeof the invention (MZ623) is described in Example 2.

The preparation of a further representative pateamine A amide derivativeof the invention (MZ757) is described in Example 5.

The preparation of a representative pateamine A carbamate derivative ofthe invention (MZ578) is described in Example 3.

The preparation of a another representative pateamine A carbamatederivative of the invention (MZ756) is described in Example 5.

In another embodiment, the invention provides pateamine A derivativeshaving formula (V), stereoisomers, racemates, and pharmaceutically saltsthereof:

wherein

Z is selected from R and OR¹, wherein R and R¹ are as described abovefor formulae (I)-(IV).

In certain embodiments, the invention provides pateamine A derivativeshaving formula (VIA) and pharmaceutically salts thereof:

wherein Z is as described above for formula (V).

In other embodiments, the invention provides pateamine A derivativeshaving formula (VIB) and pharmaceutically salts thereof:

wherein Z is as described above for formula (V).

Representative salts of the invention have formula (VII):

wherein Z is as described above for formulae (V) and A⁻ is apharmaceutically acceptable counter ion. Suitable counter ions includechloride, bromide, iodide, sulfate, phosphate, formate, acetate,trifluoroacetate, maleate, fumarate, succinate, tartrate, oxalate,citrate, malate, benzoate, toluenesulfonate, methanesulfonate, andbenzenesulfonate.

In certain embodiments, representative salts of the invention haveformula (VIIIA):

wherein Z is as described above for formula (V) and A⁻ is as describedabove for formula (VII).

In other embodiments, representative salts of the invention have formula(VIIIB):

wherein Z is as described above for formula (V) and A⁻ is as describedabove for formula (VII).

The invention provides compounds of formulae (V), (VIA), (VIB), (VII),(VIIIA), and (VIIIB) including the following embodiments:

Z is C1-C6 alkyl (e.g., methyl);

Z is C1-C6 haloalkyl (e.g., trifluoromethyl);

Z is C6-C10 aryl (e.g., phenyl); and

Z is C3-C12 alkyl in which one or more carbons are replaced with O or Natoms (e.g., —CH₂—O—CH₃, —CH₂CH₂—O—CH₃, and —CH₂CH₂—O—CH₂CH₂—O—CH₃, or—CH₂—NH—CH₃, —CH₂—N(CH₃)₂, —CH₂CH₂—NH—CH₃, and —CH₂CH₂—N(CH₃)₂).

The α-amino pateamine derivatives of the invention (i.e. compounds offormulae (I)-(VIII)) can be prepared by reacting the 2-amino group(i.e., α-amino group) of the macrocycle with a suitably reactive reagent(e.g., N-acylating or N-sulfonating reagent) to provide a variety ofα-amino pateamine derivatives.

The preparation of a representative pateamine A sulfonamide derivativeof the invention (MZ624) is described in Example 4.

Antibody Conjugates

In another aspect, the invention provides antibody drug conjugates forthe delivery of the α-amino pateamine derivatives of the invention. Theantibody drug conjugates are readily prepared from the α-amino pateaminederivatives by conjugation chemistry known in the art. In certainembodiments, the α-amino pateamine derivatives are conjugated to theantibody through the 2-amino group (i.e., α-amino group).

In certain embodiments, the antibody drug conjugate is anAntibody-Linker-Drug conjugate of the formula: Ab-(LU-D)_(p) or apharmaceutically acceptable salt or solvate thereof wherein, Ab is anantibody unit, LU is a linker unit, D is a drug unit, and p is aninteger from 1 to about 20. For the antibody drug conjugates, D includesa β-amino pateamine derivative of the invention.

Suitable antibodies include, for example, monoclonal antibodies, such aschimeric, humanized or human antibodies or an antigen-binding fragmentthereof. In some embodiments, the antibody unit comprises anantigen-binding region that binds to a target antigen.

In some embodiments, a substantial amount of the drug unit is notcleaved from the conjugate until the conjugate enters a cell with acell-surface receptor specific for the antibody unit, and the drug unitis cleaved from the antibody unit when the conjugate enters the cell. Insome embodiments, a substantial amount of the linker-drug unit is notcleaved from the conjugate until the conjugate enters a cell with acell-surface receptor specific for the antibody unit, and thelinker-drug unit is cleaved from the antibody unit when the conjugateenters the cell.

The term “antibody” as used herein, refers to a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule (i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including, but notlimited to, cancer cells. The antibody can be of any class (e.g., IgG,IgE, IgM, IgD, and IgA), or subclass (e.g., IgG1, IgG2, IgG3, IgG4,IgA1, and IgA2) of immunoglobulin molecule.

The antibody can be derived from any species. In one aspect, theantibody is of human, murine, or rabbit origin. In another aspect, theantibody is polyclonal, monoclonal, bispecific, multispecific, human,humanized or a chimeric antibody, or an epitope-binding fragment of anyof the above which immunospecifically bind to a target antigen.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies(i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts). Monoclonal antibodies are highly specific, beingdirected against a single antigenic site.

Monoclonal antibodies specifically include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, that exhibit the desired biologicalactivity. For example, a chimeric antibody may be derived from thevariable region from a mouse antibody and the constant region from ahuman antibody.

An “antibody fragment” refers to a portion of an intact antibody,typically comprising the antigen-binding or variable region thereof.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; linear antibodies; single-chain antibody molecules; an scFv;an IgG ΔCH2, a minibody, a diabody, a triabody, a tetrabody, a dsFv; ansc-Fv-Fc; an (scFv)2; a fragment produced by a Fab expression library;an anti-idiotypic (anti-Id) antibody; and multispecific antibodiesformed from antibody fragment(s).

An “intact” antibody is one which comprises an antigen-binding variableregion as well as a light chain constant domain (CO and heavy chainconstant domains, CH1, CH2 and CH3. The constant domains may be nativesequence constant domains (e.g., human native sequence constant domains)or amino acid sequence variants thereof.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(VH) connected to a variable light domain (VL) in the same polypeptidechain. By using a linker that is too short to allow pairing between thetwo domains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. “Humanized” forms of non-human (e.g., rodent) antibodies arechimeric antibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody.

These modifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

Useful monoclonal antibodies include, but are not limited to, humanmonoclonal antibodies, humanized monoclonal antibodies, antibodyfragments, or chimeric (e.g., human-mouse or other species) monoclonalantibodies. Human monoclonal antibodies may be made by any of numeroustechniques known in the art.

The antibody can also be a multispecific antibody, such as a bispecificantibody. Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of 10 different antibodymolecules.

Pharmaceutical Compositions

In another aspect, the invention provides pharmaceutical compositionsthat include a compound of the invention (i.e., a compounds of formulae(I), (II), (III), (IV), (V), (VI), (VII), or (VIII)) and apharmaceutically acceptable carrier.

The invention also provides pharmaceutical compositions that include anantibody conjugate of the invention (i.e., an antibody conjugate thatdelivers a compound of formulae (I), (II), (III), (IV), (V), (VI),(VII), or (VIII)) and a pharmaceutically acceptable carrier.

Suitable carriers include those suitable for administration to an animal(e.g., a human subject). Pharmaceutical compositions suitable forinjectable use include sterile aqueous solutions (e.g., saline,dextrose) and dispersions.

The compounds and compositions of the invention can be orallyadministered, for example, with an inert diluent or carrier, enclosed inhard or soft shell gelatin capsule, or compressed into tablets. For oraltherapeutic administration, the compounds and compositions can becombined with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The amount of active compounds in such therapeuticallyuseful compositions is such that a suitable dosage is obtained.

The compounds and compositions of the invention can be administeredparenterally or intraperitoneally. Solutions of the compounds as freebase or pharmacologically acceptable salts can be prepared in watersuitably mixed with additives, such as surfactants. Dispersions can alsobe prepared in in oils.

In the methods of the invention, the term “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result, such asreduced levels of rod gene expression or their protein products. Atherapeutically effective amount of a compound may vary according tofactors such as the disease state, age, sex, and weight of the subject,and the ability of the compound to elicit a desired response in thesubject. Dosage regimens can be adjusted to provide the optimumtherapeutic response. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the administered compound areoutweighed by the therapeutically beneficial effects.

It is to be noted that dosage values can vary with the severity of thecondition to be alleviated. For any particular subject, specific dosageregimens can be adjusted over time according to the individual need andthe professional judgment of the person administering or supervising theadministration of the compositions. Dosage ranges set forth herein areexemplary only and do not limit the dosage ranges that can be selectedby a medical practitioner. The amount of active compound in thecomposition can vary according to factors such as the disease state,age, sex, and weight of the subject. Dosage regimens can be adjusted toprovide the optimum therapeutic response. For example, a single boluscan be administered, several divided doses can be administered over timeor the dose can be proportionally reduced or increased as indicated bythe exigencies of the therapeutic situation.

In the methods, the administration of the compound can be systemicadministration to the subject. The term “subject” is intended to includemammalian organisms. Examples of subjects include humans and non-humanmammals. In specific embodiments of the invention, the subject is ahuman.

The terms “administering,” “contacting,” or “treating” include anymethod of delivery of a compounds or a pharmaceutical compositioncomprising the compound into a subject's system.

Methods of Use

In certain embodiments, the α-amino pateamine compounds of the inventionretain the advantageous potency of pateamine A (e.g., inhibitoryactivity against chronic lymphocytic leukemia (CLL) cells) and haveimproved bioavailability compared to pateamine A (e.g., lower plasmaprotein binding (PPB) in human plasma).

In one aspect, the invention provides a method for inhibiting growth ofchronic lymphocytic leukemia (CLL) cells. In the method, growth of CLLcells is inhibited by contacting CLL cells with an α-amino pateaminecompound of the invention. In certain embodiments, the method iseffective for inhibiting growth of chronic lymphocytic leukemia (CLL)cells in a subject (e.g., a human subject).

In another aspect, the invention provides a method for treating chroniclymphocytic leukemia (CLL). In the method, CLL is treated byadministering an effective amount of an α-amino pateamine compound ofthe invention to a subject (e.g., a human subject) in need thereof.

Biological Activity of 2-Amino Pateamine Derivatives Peripheral bloodfrom the CLL patients were collected in heparin vacutainer tubes andcentrifuged at 1500 rpm for 15 min to separate the plasma. The plasma(upper layer) was removed and saved for cell culture. The lower layerwas diluted with phosphate-buffered saline (PBS), and the mononuclearcells were isolated by Ficoll density-gradient centrifugation. Theisolated CLL cells were cultured at 1×10⁷ cells/mL in RPMI 1640 mediumcontaining 10% of fetal bovine serum, 10% autologous plasma, or 50%autologous plasma. Cell death in CLL lymphocytes was evaluated by flowcytometry analysis using annexin V and propidium iodide (PI) doublestaining. After incubation with increasing concentrations of PatAanalogs for 24 hours, CLL cells (1×10⁶ cells) were stained in 100 μLbinding buffer with 5 μL annexin-Cy5, and incubated for 15 minutes indark at room temperature. After staining, 300 μL binding buffer with 5μL of 50 μg/mL propidium iodide were added to each tube. Samples wereanalyzed immediately by flow cytometer. Cells stained positive foreither annexin V or PI were considered dead cells. The IC₅₀ values ofCLL killing were determined by non-linear fitting of the survival curveby the GraphPad Prism software.

TABLE 1 IC₅₀ (μM) Values CLL Assay for Representative PateamineDerivatives. Compound 10% FBS 10% plasma 50% plasma PatA 0.15 0.16 0.47DMDA Pat A 0.71 5.37 >10 MZ554 >10 >10 — MZ568 >10 >10 — MZ569a >10 >10— MZ569b >10 >10 — MZ576 3.2 6.69 — MZ578 0.83 2.01 8.12 MZ579 0.55 1.797.18 MZ577 3.01 6.36 — MZ623 0.31 0.46 — MZ624 0.37 0.65 — MZ756 1.895.61 — MZ757 <0.05 0.45 —

FIGS. 2A-2D compare IC₅₀ dose response curves (% survival of chroniclymphocytic leukemia (CLL) cells as a function of agent concentration(μM)) for pateamine A (PatA) (2A), desmethyl desamino pateamine A(DMDAPatA) (2B), α-amino pateamine A derivative (MZ578) (2C), andα-amino pateamine A derivative (MZ579) (2D) in 10% fetal bovine serum(FBS), 10% human plasma, and 50% human plasma.

The following examples are provided for the purpose of illustrating, notlimiting the invention.

EXAMPLES General Methods

All reactions were carried out under nitrogen atmosphere in flame-driedglassware. Acetonitrile, dichloromethane, methanol, and tetrahydrofuranwere purified by passage through activated molecular sieves or alumina(solvent system). Dimethylformamide (DMF) was purchased and dried over 4Å molecular sieves. All commercial reagents were used as received. ¹HNMR spectra were recorded on INOVA-500 or on Brucker-600. ¹H NMRchemical shifts are reported as 6 values in ppm relative to CDCl₃ (7.26ppm), coupling constants (J) are reported in Hertz (Hz), andmultiplicity follows convention. Flash column chromatography wasperformed using 60 Å Silica Gel as a stationary phase using a gradientsolvent system (EtOAc/hexanes as eluent unless specified otherwise).MPLC was performed using CombiFlash® Rf 200i (EtOAc/hexanes as eluent).Purification by prep-HPLC was performed on the Agilent 1260 InfinityPreparative-Scale Purification System using a Gemini HPLC column (C18, 5micron, 100×21.20 mm). Mass spectra were obtained at the center forChemical Characterization and Analysis (Texas A&M University). Thinlayer chromatography (TLC) was performed using glass-backed silica gel60F₂₅₄.

Example 1 The Preparation of a Representative Pateamine A AmideDerivative

In this example, the preparation of a representative pateamine A amidederivative of the invention,N-((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)-2,2,2-trifluoroacetamide(MZ579), is described. The preparation is illustrated schematically inFIG. 1.

(S)-4-Isopropylthiazolidine-2-thione (MZ465)

The following procedure gives a higher yield than that reported in theliterature. Erik Gálvez, P. R., Felix Urp. (2009) Preparation of(S)-4-Isopropyl-N-Propanoyl-1,3-Thiazolidine-2-Thione, Organic Syntheses86, 70. A sealed tube was charged with a solution of KOH (2.7 g, 48.4mmol, 5 equiv.) in 8 mL of water, 2 mL of EtOH, CS₂ (2.9 mL, 48.4 mmol,5 equiv.), and (S)-(+)-2-amino-3-methyl-1-butanol (1.0 g, 9.69 mmol, 1equiv.). The mixture was heated at 80° C. for 15 hours, purged with N₂to remove the excess CS₂, and neutralized by adding aqueous HCl solution(1 M). After extraction with EtOAc (3×60 mL) the organic layer waswashed with brine (5 mL), dried over MgSO₄, and concentrated in vacuo.The residue was purified on a silica gel chromatography to give thedesired product as a yellow solid (1.19 g, 76%). The characterizationdata match with those in the literature.

The compound was synthesized from MZ465 using the previously reportedprocedure. Romo, D., Rzasa, R. M., Shea, H. A., Park, K., Langenhan, J.M., Sun, L., Akhiezer, A., and Liu, J. O. (1998) Total Synthesis andImmunosuppressive Activity of (−)-Pateamine A and Related Compounds:Implementation of a β-Lactam-Based Macrocyclization, J. Am. Chem. Soc.120, 12237-12254; Low, W. K., Li, J., Zhu, M., Kommaraju, S. S.,Shah-Mittal, J., Hull, K., Liu, J. O., and Romo, D. (2014)Second-generation derivatives of the eukaryotic translation initiationinhibitor pateamine A targeting eIF4 Å as potential anticancer agents,Bioorg. Med. Chem. 22, 116-125.

2,2,2-Trichloroethyl-(S)-2-(tert-butoxycarbonylamino)pent-4-enoate(MZ434)

A mixture of (S)-2-(tert-butoxycarbonylamino)pent-4-enoic acid (10.3 g,47.9 mmol, 1 equiv.), 2,2,2-trichloroethanol (4.62 mL, 47.9 mmol, 1equiv.), DMAP (585 mg, 4.79 mmol, 0.1 equiv.), and DCC (10.9 g, 52.7mmol, 1.1 equiv.) in 200 mL of THF was stirred at 20° C. under N₂ for 24hours. The suspension was filtered by a sintered Buchner glass funneland the precipitates were washed with 20 mL of methyl tert-butyl ether.After concentration in vacuo the residue was purified on a silica gelchromatography (hexanes:methyl tert-butyl ether=30:1→20:1) to give theproduct as a colorless oil (13.4 g, 81%). ¹H NMR (500 MHz, CDCl₃) δ5.81-5.67 (m, 1H), 5.23-5.19 (m, 2H), 5.17-5.16 (m, 1H), 4.99 (d, J=7.3Hz, 1H), 4.91 (d, J=12.0 Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.54-4.48 (m,1H), 2.69-2.53 (m, 1H), 1.45 (s, 9H). HRMS (ESI⁺): Calcd. ForC₁₂H₁₉C₁₃NO₄ ([M+H]⁺), 346.0380. Found: 346.0371.

2,2,2-Trichloroethyl-(S,E)-2-(tert-butoxycarbonylamino)-6-oxohept-4-enoate(MZ456)

An ozone stream was bubbled through the stirred solution of MZ434 (1.54g, 4.44 mmol, 1 equiv.) in 50 mL of DCM at −78° C. for ca. 10 minutesuntil the color turned blue. The cold bath was removed and the solutionwas flushed with N₂ for 30 minutes. Dimethyl sulfide (2.00 mL, 27.0mmol, 6.1 equiv.) was added and the solution was stirred at 20° C. for20 hours. After concentration 1-(triphenylphosphoranylidene)-2-propanone(3.54 g, 11.1 mmol, 2.5 equiv.) and 40 mL of THF was added. The mixturewas continued to stir at 20° C. for 15 hours followed by concentration.The residue was purified on a silica gel chromatography (hexanes:methyltert-butyl ether=5:1→3:1) to give the product as a colorless oil (0.94g, 57%). ¹H NMR (500 MHz, CDCl₃) δ 6.70 (dt, J=15.9 Hz, J=7.3 Hz, 1H),6.14 (d, J=15.9 Hz, 1H), 5.13-0.509 (m, 1H), 4.92 (d, J=12.0 Hz, 1H),4.67 (d, J=12.0 Hz, 1H), 4.65-4.61 (m, 1H), 2.89-2.83 (m, 1H), 2.70-2.64(m, 1H), 2.24 (s, 3H), 1.43 (s, 9H). HRMS (ESI⁺): Calcd. ForC₁₄H₂₁C₁₃NO₅ ([M+H]⁺), 388.0485. Found: 388.0481.

(S)-2,2,2-Trichloroethyl-2-(tert-butoxycarbonylamino)-6-oxoheptanoate(MZ442)

A round-bottom flask was charged with MZ456 (6.80 g, 17.5 mmol, 1equiv.), Pd/C (10 w/w %, 800 mg, 0.752 mmol, 0.043 equiv.), EtOAc (54mL), and EtOH (6 mL) followed by setting a H₂ balloon on the top of theflask. The mixture was stirred under the H₂ atmosphere at 20° C. for 15hours and then filtered through a short celite pad. The solution wasconcentrated and the crude residue was purified on a silica gelchromatography (hexanes:methyl tert-butyl ether=3:1) to provide theproduct as a colorless oil (5.70 g, 82%). ¹H NMR (500 MHz, CDCl₃) δ 5.07(d, J=8.3 Hz, 1H), 4.91 (d, J=11.9 Hz, 1H), 4.64 (d, J=11.9 Hz, 1H),4.43-4.38 (m, 1H), 2.55-2.44 (m, 2H), 2.13 (s, 3H), 1.92-1.84 (m, 1H),1.74-1.65 (m, 3H), 1.44 (s, 9H). HRMS (ESI⁺): Calcd. For C₁₄H₂₃C₁₃NO₅([M+H]⁺), 390.0642. Found: 390.0653.

(S)-2,2,2-Trichloroethyl-7-bromo-2-(tert-butoxycarbonylamino)-6-oxoheptanoate(MZ463)

n-Butyllithium solution (2.5 M in THF, 12.8 mL, 32.0 mmol, 3 equiv.) wasslowly added to a solution of 1,1,1,3,3,3-hexamethyldisilazane (6.79 mL,32.0 mmol, 3 equiv.) in 200 mL of THF at −78° C. under N₂. The solutionwas stirred at 0° C. for 20 minutes, cooled to −78° C. andchlorotrimethylsilane (13.6 mL, 106.5 mmol, 10 equiv.) was slowly addedfollowed by MZ442 (3.99 g, 10.65 mmol, 1 equiv.) as a THF solution (20mL). After 20 minutes anhydrous triethylamine (30 mL, 213 mmol, 20equiv.) was slowly added. The mixture was continued to stir for 5minutes and the reaction was quenched with 50 mL of saturated aqueousNaHCO₃ solution and extracted with methyl tert-butyl ether (3×100 mL).The organic layer was washed with brine (5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was dissolved in 10 mL of THF and thesolution was added to a stirred suspension of N-bromosuccinimide (1.78g, 10.0 mmol, 0.94 equiv.) and NaHCO₃ (1.01 g, 12.0 mmol, 1.1 equiv.) inTHF (150 mL) at −78° C. under N₂. After stirring for 1.5 hour, thereaction was quenched by adding 30 mL of saturated aqueous NaHCO₃solution and the mixture was extracted with methyl tert-butyl ether(3×50 mL). The organic layer was washed with brine (5 mL), dried overMgSO₄ and concentrated. The crude residue was purified on a silica gelchromatography (hexanes:methyl tert-butyl ether=10:1→7:1) to provide theproduct as a yellow oil (3.29 g, 68%). ¹H NMR (500 MHz, CDCl₃) δ 5.05(d, J=8.3 Hz, 1H), 4.92 (d, J=12.0 Hz, 1H), 4.65 (d, J=12.0 Hz, 1H),4.45-4.41 (m, 1H), 3.87 (s, 2H), 2.75-2.71 (m, 2H), 1.95-1.89 (m, 1H),1.80-1.70 (m, 3H), 1.45 (s, 9H). HRMS (ESI⁺): Calcd. For C₁₄H₂₂BrCl₃NO₅([M+H]⁺), 467.9747. Found: 467.9772.

2,2,2-Trichloroethyl-(S)-5-(2-((R,E)-4-bromo-2-((triisopropylsilyl)oxy)pent-3-en-1-yl)thiazol-4-yl)-2-((tert-butoxycarbonyl)amino)pentanoate(MZ526b)

Following the same procedure as in the literature, (Romo, D., Rzasa, R.M., Shea, H. A., Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., andLiu, J. O. (1998) Total Synthesis and Immunosuppressive Activity of(−)-Pateamine A and Related Compounds: Implementation of aβ-Lactam-Based Macrocyclization, J. Am. Chem. Soc. 120, 12237-12254) thereaction between MZ463 (2.26 g, 4.81 mmol, 1.3 equiv.) and Fragment A(1.41 g, 3.70 mmol, 1 equiv.) formed the product as a colorless oil(2.08 g, 75%). ¹H NMR (500 MHz, CDCl₃) δ 6.77 (s, 1H), 5.89-5.84 (m,1H), 5.10 (d, J=8.3 Hz, 1H), 4.92 (d, J=12 Hz, 1H), 4.77 (dt, J=8.8 Hz,J=6.1 Hz, 1H), 4.62 (d, J=12 Hz, 1H), 4.48-4.42 (m, 1H), 3.24 (dd,J=14.3 Hz, J=6.4 Hz, 1H), 3.11 (dd, J=14.3 Hz, J=6.2 Hz, 1H), 2.84-2.69(m, 2H), 2.10 (s, 3H), 2.02-1.90 (m, 1H), 1.88-1.68 (m, 2H), 1.67-1.60(m, 1H), 1.44 (s, 9H), 1.02 (s, 21H). HRMS (ESI⁺): Calcd. ForC₂₉H₄₉BrCl₃N₂O₅SSi ([M+H]⁺), 749.1380. Found: 749.1349.

2,2,2-Trichloroethyl-(S)-5-(2-((R,E)-4-bromo-2-hydroxypent-3-en-1-yl)thiazol-4-yl)-2-(tert-butoxycarbonylamino)pentanoate(MZ527)

To a solution of MZ526b (2.29 g, 3.05 mmol, 1.0 equiv.) in 150 mL of THFat −20° C. under N₂ was added a pre-mixed solution of tetrabutylammoniumfluoride (1 M in THF, 9.16 mL, 9.16 mmol, 3 equiv.) and acetic acid (423μL, 7.33 mmol, 2.4 equiv.) under N₂. The mixture was kept in a −20° C.freezer for 15 hours, diluted with 200 mL of methyl tert-butyl ether,washed with saturated aqueous NaHCO₃ solution (5 mL), water (5 mL) andbrine (5 mL). The organic layer was dried over MgSO₄ and concentrated invacuo. The crude residue was purified by MPLC (hexanes:EtOAc=1:1) togive the product as a colorless oil (1.55 g, 85%). ¹H NMR (500 MHz,CDCl₃) δ 6.80 (s, 1H), 5.95 (d, J=8.7 Hz, 1H), 5.10 (d, J=8.1 Hz, 1H),4.92 (d, J=12.0 Hz, 1H), 4.75-4.71 (m, 1H), 4.63 (d, J=12.0 Hz, 1H),4.46-4.42 (m, 1H), 3.13 (d, J=5.8 Hz, 2H), 2.82 (dd, J=14.9 Hz, J=7.7Hz, 1H), 2.76 (dd, J=14.9 Hz, J=7.6 Hz, 1H), 2.29 (s, 3H), 1.98-1.91 (m,1H), 1.86-1.80 (m, 2H), 1.77-1.70 (m, 1H), 1.44 (s, 9H). OH notobserved. HRMS (ESI⁺): Calcd. For C₂₀H₂₉BrCl₃N₂O₅S ([M+H]⁺), 593.0046.Found: 593.0064.

(S)-Triisopropyl(pent-4-yn-2-yloxy)silane (OR-IV-19)

(S)-propylene oxide (5 g, 86.09 mmol, 1 equiv.) was dissolved inanhydrous THF (43 mL) and then cooled to 4° C. A solution of lithiumacetylide-ethylene diamine complex (9.51 g, 103.31 mmol, 1.2 equiv.) inDMSO (103 mL) was then added dropwise. The reaction mixture was stirredat 22° C. for 36 h and then poured into ice water (150 mL). The mixturewas extracted with diethyl ether (4×150 mL), the combined organicfractions were washed with a saturated aqueous solution of ammoniumchloride (2×150 mL), dried over sodium sulfate and concentrated underreduced pressure. The residue was dissolved in dichloromethane (100 mL),imidazole (6.45 g, 94.68 mmol, 1.1 equiv.) was added, followed bytriisopropylsilyl chloride (18.25 g, 94.68 mmol, 1.1 equiv.). Thereaction mixture was stirred at 22° C. for 12 h and then quenched by theaddition of a saturated aqueous solution of sodium bicarbonate (100 mL).The organic fraction was washed with water (100 mL), dried over sodiumsulfate and concentrated under reduced pressure. The residue waspurified by MPLC on silica gel using a gradient of hexanes and ethylacetate (9:1→1:1) to give the desired product as a colorless oil (10.83g, 52%). ¹H NMR (500 MHz, CDCl₃) δ 4.10-4.04 (m, 1H), 2.43 (ddd, J=16.5,3.5, 2.5 Hz, 1H), 2.27 (ddd, J=16.5, 8.0, 2.5 Hz, 1H), 1.98 (t, J=2.5Hz, 1H), 1.30 (d, J=6.0 Hz, 3H), 1.07-1.04 (m, 21H). LRMS (APCI⁺):Calcd. For C₁₄H₂₈OSi ([M]⁺), 240.19. Found: 240.84.

Ethyl (S,E)-5-methyl-7-((triisopropylsilyl)oxy)oct-4-en-2-ynoate(OR-IV-23)

Bis(cyclopentadienyl)-zirconium dichloride (12.16 g, 41.59 mmol, 1equiv.) was suspended in dichloroethane (100 mL) and then cooled to 0°C. A solution of trimethylaluminum (41.59 mL, 83.17 mmol, 2.0 M intoluene, 2 equiv.) was added, followed by(S)-triisopropyl(pent-4-yn-2-yloxy)silane (OR-IV-19, 10 g, 41.59 mmol, 1equiv.) dissolved in dichloroethane (20 mL). The reaction mixture wasstirred at 22° C. for 12 h, the dichloroethane and excesstrimethylaluminum were then removed by evaporation under reducedpressure at 40° C. and anhydrous THF (50 mL) was added. Separately, zincbromide (10.30 g, 45.75 mmol, 1.1 equiv.) was flame dried and dissolvedin anhydrous THF (45.75 mL). The zinc bromide solution was cannulatedinto the organoaluminum mixture at 0° C. and then the reaction mixturewas stirred at 22° C. for 30 minutes. In another flask,tris(dibenzylideneacetone)-dipalladium(0)-chloroform adduct (1.08 g,1.04 mmol, 0.025 equiv.) was dissolved in anhydrous THF (50 mL) and thentri(2-furyl)phosphine (1.45 g, 6.24 mmol, 0.15 equiv.) was added at 0°C. and stirred for 10 min. Ethyl 3-iodopropyonate (10.25 g, 45.75 mmol,1.1 equiv.) was added and the resulting solution was added via cannulato the flask containing the organozinc solution. The reaction flask wasprotected from light and stirred at 22° C. for 16 h. The reaction wasquenched with water (150 mL) and then diluted with ether (100 mL). Theaqueous phase was extracted with ether (2×200 mL), the organic fractionswere combined, washed with brine (200 mL), dried over sodium sulfate,filtered and concentrated under reduced pressure. The residue waspurified by MPLC on silica gel using a gradient of hexanes and ethylacetate (9:1→1:1) to give the desired product as a light yellow oil(7.94 g, 54%). ¹H NMR (500 MHz, CDCl₃) δ 5.41 (s, 1H), 4.25 (q, J=7.0Hz, 2H), 4.15-4.09 (m, 1H), 2.41 (dd, J=13.0, 5.5 Hz, 1H), 2.25 (dd,J=13.5, 7.0 Hz, 1H), 2.01 (s, 3H), 1.32 (t, J=7.0 Hz, 3H), 1.14 (d,J=6.0 Hz, 3H), 1.07-1.05 (m, 21H). LRMS (APCI⁺): Calcd. For C₂₀H₃₆O₃Si([M]⁺), 352.24. Found: 352.33.

(S,E)-5-methyl-7-((triisopropylsilyl)oxy)oct-4-en-2-ynoic acid (FragmentF)

Ethyl (S,E)-5-methyl-7-((triisopropylsilyl)oxy)oct-4-en-2-ynoate(OR-IV-23, 5.0 g, 14.18 mmol, 1 equiv.) was dissolved in THF/H₂O (1:1v/v, 100 mL) and then lithium hydroxide monohydrate (1.19 g, 28.36 mmol,2 equiv.) was added. The reaction mixture was stirred at 22° C. for 8 hand then quenched by the addition of a hydrochloric acid solution (50mL, 2 M). The mixture was extracted with ethyl acetate (3×50 mL), theorganic fractions were combined, dried over sodium sulfate, filtered andconcentrated under reduced pressure. The residue was purified by MPLC onsilica gel using a gradient of hexanes and ethyl acetate (4:1→1:9) togive the desired product as a light yellow oil (4.325 g, 94%). ¹H NMR(500 MHz, CDCl₃) δ 10.05 (bs, 1H) 5.44 (q, J=1.0 Hz, 1H), 4.17-4.11 (m,1H), 2.42 (dd, J=14.0, 6.0 Hz, 1H), 2.27 (dd, J=14.0, 7.0 Hz, 1H), 2.03(d, J=1.0 Hz, 3H), 1.15 (d, J=6.0 Hz, 3H), 1.07-1.05 (m, 21H). HRMS(ESI): Calcd. For C₁₈H₃₁O₃Si ([M−H]⁺), 323.2047. Found: 323.2061.

(S,E)-4-Bromo-1-(4-((S)-4-(tert-butoxycarbonylamino)-5-oxo-5-(2,2,2-trichloroethoxy)pentyl)-3λ⁴-thiazol-2-yl)pent-3-en-2-yl(S,E)-5-methyl-7-(triisopropylsilyloxy)oct-4-en-2-ynoate (MZ530)

Following the same procedure as in the literature (Romo, D., Choi, N.S., Li, S., Buchler, I., Shi, Z., and Liu, J. O. (2004) Evidence forSeparate Binding and Scaffolding Domains in the Immunosuppressive andAntitumor Marine Natural Product, Pateamine A: Design, Synthesis, andActivity Studies Leading to a Potent Simplified Derivative, J. Am. Chem.Soc. 126, 10582-10588), MZ527 (950 mg, 1.60 mmol, 1 equiv.) was reactedwith Fragment F (623 mg, 1.92 mmol, 1.2 equiv.) to afford the product asa colorless oil (1.20 g, 83%). ¹H NMR (500 MHz, CDCl₃) δ 6.80 (s, 1H),5.89-5.86 (m, 1H), 5.73 (dt, J=9.6 Hz, J=6.7 Hz, 1H), 5.40 (s, 1H), 5.12(d, J=8.1 Hz, 1H), 4.92 (d, J=12.0 Hz, 1H), 4.63 (d, J=12.0 Hz, 1H),4.46-4.42 (m, 1H), 4.15-4.09 (m, 1H), 3.38 (dd, J=14.8 Hz, J=7.1 Hz,1H), 3.27 (dd, J=14.8 Hz, J=6.4 Hz, 1H), 2.83-2.72 (m, 2H), 2.41 (dd,J=13.3 Hz, J=5.4 Hz, 1H), 2.28 (d, J=1.1 Hz, 3H), 2.25 (dd, J=13.3 Hz,J=6.9 Hz, 1H), 2.01 (s, 3H), 1.98-1.91 (m, 1H), 1.87-1.80 (m, 2H),1.78-1.71 (m, 1H), 1.44 (s, 9H), 1.13 (d, J=6.0 Hz, 3H), 1.05 (s, 21H).HRMS (ESI⁺): Calcd. For C₃₈H₅₉BrCl₃N₂O₇SSi ([M+H]⁺), 901.2041. Found:901.2099.

(S,E)-4-Bromo-1-(4-((S)-4-(tert-butoxycarbonylamino)-5-oxo-5-(2,2,2-trichloroethoxy)pentyl)thiazol-2-yl)pent-3-en-2-yl (S,E)-7-hydroxy-5-methyloct-4-en-2-ynoate(MZ533)

Tetrabutylammonium fluoride (1 M in THF, 5 mL, 5.0 mmol) was mixed withHF/py (70 w/w % HF, 138 μL, 5.3 mmol) at 0° C. and 1.69 mL of themixture was added to a solution of MZ530 (290 mg, 0.322 mmol) in THF(1.2 mL) at 0° C. The reaction was maintained at the same temperaturefor 6 hours. The mixture was diluted with 100 mL of DCM and was washedwith saturated aqueous NaHCO₃ solution (5 mL), water (5 mL), and brine(5 mL). The organic layer was dried over MgSO₄ and concentrated. Thecrude residue was purified on a silica gel chromatography to give thedesired product as a colorless oil (185 mg, 77%). ¹H NMR (500 MHz,CDCl₃) δ 6.81 (s, 1H), 5.89-5.86 (m, 1H), 5.78 (dt, J=9.4 Hz, J=6.6 Hz,1H), 5.47-5.46 (m, 1H), 5.14 (d, J=8.3 Hz, 1H), 4.92 (d, J=12.0 Hz, 1H),4.63 (d, J=12.0 Hz, 1H), 4.45-4.41 (m, 1H), 4.03-3.97 (m, 1H), 3.38 (dd,J=14.8 Hz, J=7.3 Hz, 1H), 3.27 (dd, J=14.8 Hz, J=6.2 Hz, 1H), 2.83-2.73(m, 2H), 2.33-2.25 (m, 2H), 2.29 (d, J=1.2 Hz, 3H), 2.03 (d, J=1.1 Hz,3H), 1.97-1.89 (m, 1H), 1.86-1.80 (m, 2H), 1.78-1.71 (m, 1H), 1.44 (s,9H), 1.22 (d, J=6.2 Hz, 3H). OH not observed. HRMS (ESI⁺): Calcd. ForC₂₉H₃₉BrCl₃N₂O₇S ([M+H]⁺), 743.0727. Found: 743.0755.

(S)-5-(2-((S,E)-4-Bromo-2-(((S,E)-7-hydroxy-5-methyloct-4-en-2-ynoyl)oxy)pent-3-en-1-yl)thiazol-4-yl)-2-(tert-butoxycarbonylamino)pentanoic acid (MZ536)

Following the known procedure, (Romo, D., Rzasa, R. M., Shea, H. A.,Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., and Liu, J. O. (1998)Total Synthesis and Immunosuppressive Activity of (−)-Pateamine A andRelated Compounds: Implementation of a β-Lactam-Based Macrocyclization,J. Am. Chem. Soc. 120, 12237-12254) MZ536 was synthesized from MZ533(400 mg, 0.537 mmol) as a colorless oil (241 mg, 73%). ¹H NMR (500 MHz,CDCl₃) δ 6.86 (s, 1H), 5.88-5.86 (m, 1H), 5.77-5.72 (m, 1H), 5.46 (s,1H), 5.29 (d, J=7.8 Hz, 1H), 4.35-4.31 (m, 1H), 4.05-3.99 (m, 1H), 3.44(dd, J=14.8 Hz, J=7.6 Hz, 1H), 3.35 (dd, J=14.8 Hz, J=5.7 Hz, 1H), 2.79(t, J=6.9 Hz, 2H), 2.33-2.25 (m, 2H), 2.29 (s, 3H), 2.01 (s, 3H),1.93-1.86 (m, 1H), 1.81-1.74 (m, 3H), 1.44 (s, 9H), 1.22 (d, J=6.2 Hz,3H). OH not observed. HRMS (ESI⁺): Calcd. For C₂₇H₃₈BrN₂O₇S ([M+H]⁺),613.1583. Found: 613.1568.

tert-Butyl((1²Z,3S,8E,11S,14S)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1 (2,4)-thiazolacycloheptadecaphane-6-yn-8-en-14-yl)carbamate(MZ538)

Following the known procedure, (Romo, D., Choi, N. S., Li, S., Buchler,I., Shi, Z., and Liu, J. O. (2004) Evidence for Separate Binding andScaffolding Domains in the Immunosuppressive and Antitumor MarineNatural Product, Pateamine A: Design, Synthesis, and Activity StudiesLeading to a Potent Simplified Derivative, J. Am. Chem. Soc. 126,10582-10588.) MZ536 (52 mg, 0.085 mmol) afforded the desired product asa colorless oil (31 mg, 68%). ¹H NMR (500 MHz, CDCl₃) δ 6.79 (s, 1H),6.03 (d, J=9.1 Hz, 1H), 5.93-5.87 (m, 1H), 5.40 (s, 1H), 5.27-5.21 (m,1H), 5.14 (d, J=7.7 Hz, 1H), 4.31-4.27 (m, 1H), 3.35-3.27 (m, 2H), 2.79(t, J=7.3 Hz, 2H), 2.43-2.30 (m, 2H), 2.41 (s, 3H), 1.93 (s, 3H),1.90-1.86 (m, 1H), 1.77-1.69 (m, 1H), 1.67-1.60 (m, 2H), 1.45 (s, 9H),1.29 (d, J=6.2 Hz, 3H). HRMS (ESI⁺): Calcd. For C₂₇H₃₆BrN₂O₆S ([M+H]⁺),595.1477. Found: 595.1474.

tert-Butyl-((1²Z,3S,6Z,8E,11S,14S)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)carbamate(MZ554)

A H₂ balloon was placed on the top of a flask containing MZ538 (53 mg,0.089 mmol, 1 equiv.), lindlar catalyst (25 mg), and MeOH (10 mL). Themixture was stirred at 20° C. for 2.5 hours until no starting materialvisible on TLC and was filtered through a cotton pad which was rinsedwith 5 mL of EtOAc. The solvents were evaporated in vacuo and the cruderesidue was purified on a silica gel chromatography to provide theproduct as a colorless oil (50 mg, 94%). ¹H NMR (500 MHz, CDCl₃) δ 6.94(d, J=12.0 Hz, 1H), 6.78 (s, 1H), 6.69 (t, J=11.6 Hz, 1H), 6.06 (td,J=9.7 Hz, J=4.1 Hz, 1H), 6.00-5.98 (m, 1H), 5.45 (d, J=11.5 Hz, 1H),5.29-5.23 (m, 1H), 4.93 (d, J=8.9 Hz, 1H), 4.21-4.16 (m, 1H), 3.25-3.15(m, 2H), 2.86-2.81 (m, 1H), 2.71-2.65 (m, 1H), 2.43 (d, J=1.1 Hz, 3H),2.40 (dd, J=13.5 Hz, J=11.2 Hz, 1H), 2.23 (d, J=13.5 Hz, 1H), 1.94-1.82(m, 3H), 1.81 (s, 3H), 1.73-1.66 (m, 1H), 1.45 (s, 9H), 1.29 (d, J=6.3Hz, 3H). HRMS (ESI⁺): Calcd. For C₂₇H₃₈BrN₂O₆S ([M+H]⁺), 597.1634.Found: 597.1653.

(1²Z,3S,6Z,8E,11S,14S)-14-Amino-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-diene-5,13-dione(MZ556)

A solution of trifluoroacetic acid (0.4 mL) in DCM (1.6 mL) was cooledto 0° C. and added to MZ554 (36 mg, 0.060 mmol) at 0° C. under N₂. Thereaction was kept in a 4° C. refrigerator for 15 hours, and the solventswere evaporated while the flask was kept at 0° C. The crude residue waspurified on a silica gel chromatography (dichloromethane:MeOH=20:1) togive the product as a colorless oil in the form of a TFA salt (35 mg,98%). ¹H NMR (500 MHz, CDCl₃) δ 6.94 (d, J=10.9 Hz, 1H), 6.87 (s, 1H),6.73 (t, J=11.3 Hz, 1H), 6.00 (brs, 2H), 5.51 (d, J=11.3 Hz, 1H), 5.35(brs, 1H), 3.85 (brs, 1H), 3.21 (brs, 2H), 2.80 (brs, 1H), 2.68 (brs,1H), 2.46 (d, J=12.6 Hz, 1H), 2.39 (s, 3H), 2.28 (d, J=12.6 Hz, 1H),1.91-1.81 (m, 3H), 1.77 (s, 3H), 1.73-1.64 (m, 1H), 1.28 (d, J=4.7 Hz,3H). NH₂ not observed. HRMS (ESI⁺): Calcd. For C₂₂H₃₀BrN₂O₄S ([M+H]⁺),497.1110. Found: 497.1117.

1,1,1-Trichloro-2-methylpropan-2-yl((1²Z,3S,6Z,8E,11S,14S)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-1⁴-yl)carbamate (MZ569a)

Following the known procedure, (Romo, D., Rzasa, R. M., Shea, H. A.,Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., and Liu, J. O. (1998)Total Synthesis and Immunosuppressive Activity of (−)-Pateamine A andRelated Compounds: Implementation of a β-Lactam-Based Macrocyclization,J. Am. Chem. Soc. 120, 12237-12254) MZ556 (4.5 mg, 0.0074 mmol) affordedthe desired product as a colorless oil (3.7 mg, 71%). ¹H NMR (500 MHz,CDCl₃) δ 6.95 (d, J=11.8 Hz, 1H), 6.78 (s, 1H), 6.70 (t, J=11.6 Hz, 1H),6.07 (td, J=9.4 Hz, J=4.6 Hz, 1H), 6.00-5.97 (m, 1H), 5.45 (d, J=11.5Hz, 1H), 5.29 (d, J=8.6 Hz, 1H), 5.28-5.23 (m, 1H), 4.18 (ddd, J=9.7 Hz,J=9.3 Hz, J=4.4 Hz, 1H), 3.21-3.17 (m, 2H), 2.88-2.82 (m, 1H), 2.69(ddd, J=14.9 Hz, J=9.0 Hz, J=5.1 Hz, 1H), 2.42 (s, 3H), 2.40 (dd, J=13.6Hz, J=11.1 Hz, 1H), 2.23 (d, J=13.6 Hz, 1H), 1.93 (s, 3H), 1.90 (s, 3H),1.82 (s, 3H), 1.76-1.69 (m, 2H), 1.63-1.58 (m, 1H), 1.51-1.45 (m, 1H),1.29 (d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. For C₂₇H₃₅BrCl₃N₂O₆S([M+H]⁺), 699.0465. Found: 699.0427.

N-((1²Z,3S,6Z,8E,11S,14S)-3-((E)-2-Bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-1⁴-yl)-2,2,2-trifluoroacetamide(MZ569b)

To the solution of MZ556 (9.2 mg, 0.015 mmol, 1 equiv.) in 0.6 mL of DCMwas added pyridine (0.3 mL, in excess) at 0° C. followed bytrifluoroacetic anhydride (10.5 mL, 0.075 mmol, 5 equiv.). After 30minutes the mixture was warmed to 20° C. and continued to stir for 2hours before being concentrated. The residue was purified on a silicagel chromatography (hexanes:EtOAc=5:1) to give the product as acolorless oil (6.8 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 6.98 (d, J=11.7Hz, 1H), 6.94 (d, J=8.1 Hz, 1H), 6.78 (s, 1H), 6.73 (t, J=11.6 Hz, 1H),6.06 (td, J=9.2 Hz, J=4.2 Hz, 1H), 6.02-5.90 (m, 1H), 5.51 (d, J=11.3Hz, 1H), 5.29-5.23 (m, 1H), 4.41-4.36 (m, 1H), 3.26-3.19 (m, 2H),2.88-2.82 (m, 1H), 2.67-2.62 (m, 1H), 2.43 (dd, J=13.7 Hz, J=11.4 Hz,1H), 2.42 (s, 3H), 2.23 (d, J=13.7 Hz, 1H), 1.89-1.79 (m, 3H), 1.83 (s,3H), 1.52-1.47 (m, 1H), 1.31 (d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. ForC₂₄H₂₉BrF₃N₂O₅S ([M+H]⁺), 593.0933. Found: 593.0951.

1,1,1-Trichloro-2-methylpropan-2-yl((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazol acycloheptadecaphane-6,8-dien-14-yl)carbamate (MZ576)

The coupling between MZ569a (7.0 mg, 0.010 mmol, 1 equiv.) and FragmentG (8.3 mg, 0.020 mmol, 2 equiv.) followed the known procedure. Low, W.K., Li, J., Zhu, M., Kommaraju, S. S., Shah-Mittal, J., Hull, K., Liu,J. O., and Romo, D. (2014) Second-generation derivatives of theeukaryotic translation initiation inhibitor pateamine A targeting eIF4 Åas potential anticancer agents, Bioorg. Med. Chem. 22, 116-125. Thereaction mixture was kept at 20° C. for 15 hours and transferreddirectly to a silica gel chromatography for purification(dichloromethane:MeOH:triethylamine=50:1:0.1). The product was furtherpurified by the Prep-HPLC (solvent A: H₂O buffered with 8 mM HCOOH and12 mM NH₃,H₂O, pH=9.0; solvent B: CH₃CN/H₂O (9:1 v/v) buffered with 8 mMHCOOH and 12 mM NH₃,H₂O; isocratic elution, solvent A/solvent B=1:4).The collected fractions were concentrated to give a mixture of theproduct and solid ammonium formate, upon which DCM (10 mL) was added andthe suspension was filtered through a sintered Buchner glass funnel. Theprecipitates were rinsed with extra dichloromethane (2×5 mL). Afterconcentration in vacuo the product was obtained as a colorless oil inthe form a salt with formic acid (4.5 mg, 73%). ¹H NMR (500 MHz, CDCl₃)δ 8.49 (brs, 1H), 6.97 (d, J=11.8 Hz, 1H), 6.80 (s, 1H), 6.69 (t, J=11.6Hz, 1H), 6.38 (d, J=16.0 Hz, 1H), 6.32 (d, J=16.0 Hz, 1H), 6.28 (ddd,J=10.3 Hz, J=9.0 Hz, J=3.5 Hz, 1H), 5.67 (t, J=7.6 Hz, 1H), 5.63 (d,J=8.9 Hz, 1H), 5.48 (d, J=11.4 Hz, 1H), 5.30 (d, J=8.9 Hz, 1H),5.30-5.25 (m, 1H), 4.19 (ddd, J=9.7 Hz, J=8.9 Hz, J=4.6 Hz, 1H), 3.59(d, J=7.7 Hz, 2H), 3.24 (dd, J=14.5 Hz, J=10.5 Hz, 1H), 3.19 (dd, J=14.5Hz, J=3.6 Hz, 1H), 2.89-2.83 (m, 1H), 2.69 (ddd, J=13.5 Hz, J=8.6 Hz,J=5.2 Hz, 1H), 2.60 (s, 6H), 2.42 (dd, J=13.5 Hz, J=11.5 Hz, 1H), 2.24(d, J=13.5 Hz, 1H), 1.96 (s, 3H), 1.94 (s, 3H), 1.92-1.88 (m, 1H), 1.91(s, 3H), 1.86 (s, 3H), 1.81 (s, 3H), 1.78-1.73 (m, 1H), 1.30 (d, J=6.3Hz, 3H), 1.28-1.20 (m, 2H). HRMS (ESI⁺): Calcd. For C₃₅H₄₉C₁₃N₃O₆S([M+H]⁺), 744.2408. Found: 744.2500.

(12Z,3S,6Z,8E,11S,14S)-14-Amino-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-diene-5,13-dione(MZ577)

A mixture of MZ576 (1.4 mg, 1.8 μmol), Cd—Pb couple (5.5 mg), aqueousNH₄OAc solution (1 M, 50 μL), H₂O (150 μL), and THF (200 μL) was stirredat 20° C. under N₂. After 1 hour an extra portion of Cd—Pb couple (1.4mg) was added and the mixture was continued to stir for another 30minutes. The solvents were evaporated and CHCl₃ (5 mL) was added to theresidue. The suspension was filtered through a sintered Buchner glassfunnel and the solvent was concentrated. The crude residue was purifiedby prep-HPLC following the same procedure as in MZ576 to give theproduct in the form of a salt with formic acid (0.5 mg, 47%). ¹H NMR(500 MHz, CDCl₃) δ 8.44 (brs, 1H), 6.98 (d, J=11.7 Hz, 1H), 6.77 (s,1H), 6.69 (t, J=11.6 Hz, 1H), 6.38 (d, J=15.9 Hz, 1H), 6.32 (d, J=16.0Hz, 1H), 6.27 (td, J=8.9 Hz, J=5.2 Hz, 1H), 5.67 (t, J=7.6 Hz, 1H), 5.63(d, J=8.8 Hz, 1H), 5.46 (d, J=11.5 Hz, 1H), 5.27-5.21 (m, 1H), 3.55 (d,J=7.7 Hz, 2H), 4.82 (q, J=4.7 Hz, 1H), 3.23-3.19 (m, 2H), 2.88-2.82 (m,1H), 2.73 (ddd, J=13.9 Hz, J=8.2 Hz, J=5.1 Hz, 1H), 2.57 (s, 6H), 2.42(dd, J=13.5 Hz, J=11.0 Hz, 1H), 2.22 (d, J=13.5 Hz, 1H), 1.97 (s, 3H),1.94-1.88 (m, 1H), 1.86 (s, 3H), 1.82 (s, 3H), 1.68-1.61 (m, 1H), 1.28(d, J=6.3 Hz, 3H), 1.23-1.15 (m, 2H). NH₂ not observed. HRMS (ESI):Calcd. For C₃₀H₄₄N₃O₄S ([M+H]⁺), 542.3053. Found: 542.3097.

N-((1Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)-2,2,2-trifluoroacetamide(MZ579)

The coupling between MZ569b (6.8 mg, 0.0115 mmol, 1 equiv.) and FragmentG (9.5 mg, 0.023 mmol, 2 equiv.) followed the known procedure. Low, W.K., Li, J., Zhu, M., Kommaraju, S. S., Shah-Mittal, J., Hull, K., Liu,J. O., and Romo, D. (2014) Second-generation derivatives of theeukaryotic translation initiation inhibitor pateamine A targeting eIF4 Åas potential anticancer agents, Bioorg. Med. Chem. 22, 116-125. Thepurification was the same as for MZ576. The desired product was obtainedin the form of salt with formic acid as a colorless oil (5.5 mg, 70%).¹H NMR (500 MHz, CDCl₃) δ 8.51 (brs, 1H), 7.00 (d, J=11.9, 1H),6.99 (d,J=8.3 Hz, 1H), 6.80 (s, 1H), 6.71 (t, J=11.6 Hz, 1H), 6.39 (d, J=15.8Hz, 1H), 6.31 (d, J=15.8 Hz, 1H), 6.25 (ddd, J=10.2 Hz, J=8.9 Hz, J=3.7Hz, 1H), 5.67 (t, J=7.3 Hz, 1H), 5.64 (d, J=9.1 Hz, 1H), 5.53 (d, J=11.3Hz, 1H), 5.31-5.24 (m, 1H), 4.41-4.36 (m, 1H), 3.53 (d, J=7.6 Hz, 2H),3.25 (dd, J=14.5 Hz, J=10.2 Hz, 1H), 3.20 (dd, J=14.5 Hz, J=3.7 Hz, 1H),2.89-2.83 (m, 1H), 2.69-2.63 (m, 1H), 2.55 (s, 6H), 2.44 (dd, J=13.8 Hz,J=11.3 Hz, 1H), 2.25 (d, J=13.8 Hz, 1H), 1.95 (d, J=1.2 Hz, 3H),1.91-1.88 (m, 1H), 1.85-1.83 (m, 2H), 1.86 (s, 3H), 1.81 (s, 3H),1.56-1.48 (m, 1H), 1.31 (d, J=6.2 Hz, 3H). HRMS (ESI⁺): Calcd. ForC₃₂H₄₃F₃N₃O₅S ([M+H]⁺), 638.2876. Found: 638.2768.

Example 2 The Preparation of a Representative Pateamine A AmideDerivative

In this example, the preparation of a representative pateamine A amidederivative of the invention,N-((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)acetamide (MZ623), is described.

N-((1²Z,3S,6Z,8E,11S,14S)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl) acetamide (MZ620). Toa solution of MZ556 (6.5 mg, 0.011 mmol, 1 equiv.) in 0.4 mL of CH₂Cl₂were added pyridine (0.2 mL, excess) and AcCl (3.8 μL, 0.053 mmol, 5equiv.) at 0° C. The temperature was increased to 20° C. within 1 h andthe reaction was continued for 15 hours. The mixture was diluted with 25mL of EtOAc, washed with H₂O and brine. The organic phase was dried overMgSO₄ and concentrated in vacuo. The residue was purified by a flashchromatography (CH₂C12:MTBE=2:1) to give the desired product as acolorless oil (3.4 mg, 60%). ¹H NMR (500 MHz, CDCl₃) δ 6.94 (d, J=11.6Hz, 1H), 6.80 (s, 1H), 6.71 (t, J=11.6 Hz, 1H), 6.05 (td, J=9.7 Hz,J=3.2 Hz, 1H), 6.01 (app. d, J=9.1 Hz, 1H), 5.89 (d, J=8.7 Hz, 1H), 5.49(d, J=11.3 Hz, 1H), 5.30-5.24 (m, 1H), 4.53 (td, J=8.9 Hz, J=5.0 Hz,1H), 3.24 (dd, J=14.4 Hz, J=9.9 Hz, 1H), 3.19 (dd, J 14.4=Hz, J=2.9 Hz,1H), 2.86-2.80 (m, 1H), 2.68-2.62 (m, 1H), 2.43 (d, J=14.2 Hz, 1H), 2.41(s, 3H), 2.26 (d, J=14.2 Hz, 1H), 2.04 (s, 3H), 1.85-1.78 (m, 3H), 1.81(s, 3H), 1.73-1.66 (m, 1H), 1.29 (d, J=6.2 Hz, 3H). HRMS (ESI⁺): Calcd.For C₂₄H₃₂BrN₂O₅S ([M+H]⁺), 539.1215. Found: 539.1191.

N-((1²Z,3S,6Z,8E,11S,14S)-3-((E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)acetamide (MZ623). The coupling between MZ620 (2.0 mg, 0.0037 mmol, 1equiv.) and Fragment G (3.1 mg, 0.0074 mmol, 2 equiv.) was based on theknown procedure. Low, W. K., Li, J., Zhu, M., Kommaraju, S. S.,Shah-Mittal, J., Hull, K., Liu, J. O., and Romo, D. (2014)Second-generation derivatives of the eukaryotic translation initiationinhibitor pateamine A targeting eIF4 Å as potential anticancer agents,Bioorg. Med. Chem. 22, 116-125. The purification procedure was the sameas that of MZ576, but the product still contained certain amount ofimpurities. Further purified on prep-HPLC using a modified condition(solvent A: H₂O buffered with 8 mM HCOOH and 12 mM NH₃,H₂O, pH=9.0;solvent B: methanol; gradient elution, solvent A/solvent B=2:3-1:4within 12 minutes) gave the pure product in the form of salt with formicacid as a colorless oil (0.65 mg, 30%). ¹H NMR (500 MHz, CDCl₃) δ 8.53(brs, 1H), 6.96 (d, J=11.9 Hz, 1H), 6.82 (s, 1H), 6.69 (t, J=11.5 Hz,1H), 6.38 (d, J=15.9 Hz, 1H), 6.28 (d, J=15.9 Hz, 1H), 6.25 (ddd, J=10.8Hz, J=8.7 Hz, J=3.3 Hz, 1H), 5.91 (d, J=8.6 Hz, 1H), 5.66 (t, J=7.2 Hz,1H), 5.62 (d, J=8.7 Hz, 1H), 5.52 (d, J=11.3 Hz, 1H), 5.33-5.27 (m, 1H),4.54 (td, J=9.1 Hz, J=5.3 Hz, 1H), 3.31 (d, J=7.2 Hz, 2H), 3.27 (dd,J=14.4 Hz, J=11.0 Hz, 1H), 3.19 (dd, J=14.4 Hz, J=2.9 Hz, 1H), 2.84 (dt,J=15.3 Hz, J=6.6 Hz, 1H), 2.66 (dt, J=15.3 Hz, J=6.5 Hz, 1H), 2.43 (dd,J=13.5 Hz, J=11.3 Hz, 1H), 2.41 (s, 6H), 2.25 (d, J=13.5 Hz, 1H),2.06-1.99 (m, 3H), 2.04 (s, 3H), 1.95 (s, 3H), 1.83 (s, 3H), 1.80 (s,3H), 1.74-1.67 (m, 1H), 1.29 (d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. ForC₃₂H₄₆N₃O₅S ([M+H]⁺), 584.3158. Found: 584.3137.

Example 3 The Preparation of a Representative Pateamine a CarbamateDerivative

In this example, the preparation of a representative pateamine Acarbamate derivative of the invention, tert-Butyl((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)carbamate (MZ578), is described.

tert-Butyl((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)carbamate (MZ578). The coupling between MZ554 (6.2 mg, 0.0104 mmol, 1equiv.) and Fragment G (8.6 mg, 0.0207 mmol, 2 equiv.) followed theknown procedure. Low, W. K., Li, J., Zhu, M., Kommaraju, S. S.,Shah-Mittal, J., Hull, K., Liu, J. O., and Romo, D. (2014)Second-generation derivatives of the eukaryotic translation initiationinhibitor pateamine A targeting eIF4 Å as potential anticancer agents,Bioorg. Med. Chem. 22, 116-125. The desired product was obtained in theform of salt with formic acid as a colorless oil (4.8 mg, 67%) afterpurification by prep-HPLC using the same conditions as MZ576. ¹H NMR(500 MHz, CDCl₃) δ 8.52 (brs, 1H), 6.96 (d, J=11.7 Hz, 1H), 6.80 (s,1H), 6.68 (t, J=11.7 Hz, 1H), 6.38 (d, J=15.8 Hz, 1H), 6.30 (d, J=15.8Hz, 1H), 6.27 (ddd, J=10.8 Hz, J=9.0 Hz, J=3.4 Hz, 1H), 5.66 (t, J=7.4Hz, 1H), 5.62 (d, J=8.8 Hz, 1H), 5.48 (d, J=11.5 Hz, 1H), 5.31-5.24 (m,1H), 4.95 (d, J=8.8 Hz, 1H), 4.23-4.17 (m, 1H), 3.43 (d, J=7.6 Hz, 2H),3.25 (dd, J=14.5 Hz, J=10.6 Hz, 1H), 3.19 (dd, J=14.5 Hz, J=3.5 Hz, 1H),2.88-2.82 (m, 1H), 2.71-2.66 (m, 1H), 2.49 (s, 6H), 2.41 (dd, J=13.8 Hz,J=11.3 Hz, 1H), 2.23 (d, J=13.8 Hz, 1H), 1.96 (s, 3H), 1.92-1.87 (m,3H), 1.86 (s, 3H), 1.80 (s, 3H), 1.74-1.67 (m, 1H), 1.45 (s, 9H), 1.29(d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. For C₃₅H₅₂N₃O₆S ([M+H]⁺),642.3577. Found: 642.3496.

Example 4 The Preparation of a Representative Pateamine A SulfonamideDerivative

In this example, the preparation of a representative pateamine Asulfonamide derivative of the invention,N-((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)methyl sulfonamide (MZ624), is described.

N-((1²Z,3S,6Z,8E,11S,14S)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)methanesulfonamide(MZ622). A solution of MZ565 (4.0 mg, 0.0068 mmol, 1 equiv.) in 3 mL ofmethanol passed through an amino cartridge (BAKERBOND Spe™ AminoDisposable Extraction Column, 500 mg) which was pre-equilibrated with 5mL of methanol. The cartridge was washed with another 3 mL of methanol.The combined solutions were concentrated, to which were added CH₂Cl₂(0.4 mL), pyridine (0.2 mL) and Ms₂O (6.0 mg, 0.034 mmol, 5 equiv.) at0° C. The yellow solution was stirred at 20° C. for 2 hours andconcentrated. The residue was purified by a flash chromatography(hexanes:EtOAc=3:1) to afford MZ622 as a yellow oil (3.2 mg, 49%). ¹HNMR (500 MHz, CDCl₃) δ 6.97 (d, J=11.9 Hz, 1H), 6.79 (s, 1H), 6.68 (t,J=11.6 Hz, 1H), 6.32 (ddd, J=9.4 Hz, J=7.9 Hz, J=6.2 Hz, 1H), 5.99 (dq,J=9.3 Hz, J=1.2 Hz, 1H), 5.44 (d, J=11.5 Hz, 1H), 5.29-5.23 (m, 1H),4.71 (d, J=9.9 Hz, 1H), 3.97 (td, J=10.4 Hz, J=4.0 Hz, 1H), 3.24-3.18(m, 2H), 2.92 (s, 3H), 2.90-2.78 (m, 2H), 2.43 (s, 3H), 2.42 (dd, J=13.5Hz, J=10.8 Hz, 1H), 2.25 (d, J=13.5 Hz, 1H), 2.02-1.95 (m, 2H),1.92-1.87 (m, 1H), 1.82 (s, 3H), 1.51-1.46 (m, 1H), 1.31 (d, J=6.3 Hz,3H). HRMS (ESI⁺): Calcd. For C₂₃H₃₂BrN₂O₆S₂ ([M+H]⁺), 573.0729. Found:573.0747.

N-((1²Z,3S,6Z,8E,11S,14S)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)methanesulfonamide (MZ624). The coupling between MZ622 (3.0 mg, 0.0052mmol, 1 equiv.) and Fragment G (4.3 mg, 0.0104 mmol, 2 equiv.) followedthe known procedure. Low, W. K., Li, J., Zhu, M., Kommaraju, S. S.,Shah-Mittal, J., Hull, K., Liu, J. O., and Romo, D. (2014)Second-generation derivatives of the eukaryotic translation initiationinhibitor pateamine A targeting eIF4 Å as potential anticancer agents,Bioorg. Med. Chem. 22, 116-125. The purification was the same as forMZ576. The desired product was obtained in the form of salt with formicacid as a colorless oil (2.8 mg, 81%). ¹H NMR (500 MHz, CDCl₃) δ 8.45(brs, 1H), 6.99 (d, J=11.8 Hz, 1H), 6.81 (s, 1H), 6.67 (t, J=11.6 Hz,1H), 6.40-6.32 (m, 2H), 6.31 (td, J=9.4 Hz, J=4.3 Hz, 1H), 5.67 (t,J=7.4 Hz, 1H), 5.63 (d, J=9.1 Hz, 1H), 5.47 (d, J=11.4 Hz, 1H),5.31-5.25 (m, 1H), 4.76 (d, J=9.8 Hz, 1H), 3.98 (td, J=10.2 Hz, J=3.9Hz, 1H), 3.63-3.60 (m, 2H), 3.26-3.18 (m, 2H), 2.93 (s, 3H), 2.90-2.85(m, 1H), 2.83-2.76 (m, 1H), 2.62 (s, 6H), 2.43 (dd, J=13.5 Hz, J=11.1Hz, 1H), 2.25 (d, J=13.5 Hz, 1H), 2.02-1.97 (m, 3H), 1.97 (s, 3H), 1.87(s, 3H), 1.81 (s, 3H), 1.79-1.72 (m, 1H), 1.31 (d, J=6.3 Hz, 3H). HRMS(ESI⁺): Calcd. For C₃₁H₄₆N₃O₆S₂([M+H]⁺), 620.2828. Found: 620.2855.

Example 5 The Preparation of a Representative Pateamine A Derivatives

In this example, the preparations of representative pateamine Aderivatives of the invention,1,1,1-trichloro-2-methylpropan-2-yl((1²Z,3S,6Z,8E,11S,14R)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)carbamate (MZ756) andN-((1²Z,3S,6Z,8E,11S,14R)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)acetamide (MZ757), are described.

2,2,2-Trichloroethyl (R)-2-(tert-butoxycarbonylamino)pent-4-enoate(MZ722)

To a solution of (R)-2-(tert-butoxycarbonylamino)pent-4-enoic acid (18.5g, 86.0 mmol, 1 equiv) in 300 mL of THF at 0° C. were added DMAP (1.05mg, 8.60 mmol, 0.1 equiv.), DCC (10.9 g, 52.7 mmol, 1.1 equiv.) and2,2,2-trichloroethanol (8.29 mL, 86.0 mmol, 1 equiv.). The ice bath wasremoved after 10 minutes and the mixture was stirred at 20° C. under N₂for 24 hours. The suspension was filtered by a sintered Buchner glassfunnel and the precipitates were washed with 20 mL of methyl tert-butylether. After concentration in vacuo the residue was purified by MPLC(hexanes:ethyl acetate=90:10) to give the product as a colorless oil(24.1 g, 81%). ¹H NMR (500 MHz, CDCl₃) δ 5.74-5.66 (m, 1H), 5.17-5.13(m, 2H), 5.03 (d, J=7.6 Hz, 1H), 4.88 (d, J=12.0 Hz, 1H), 4.62 (d,J=12.0 Hz, 1H), 4.47 (q, J=6.6 Hz, 1H), 2.63-2.51 (m, 2H), 1.41 (s, 9H).HRMS (ESI⁺): Calcd. For C₁₂H₁₈C₁₃NO₄Na ([M+Na]⁺), 368.0194. Found:368.0210.

2,2,2-Trichloroethyl(R,E)-2-(tert-butoxycarbonylamino)-6-oxohept-4-enoate (MZ725)

An ozone stream was bubbled through the stirred solution of MZ722 (10.6g, 30.6 mmol, 1 equiv) in 250 mL of DCM at −78° C. for ca. 10 minutesuntil the color turned blue. The cold bath was removed and the solutionwas flushed with N₂ for 30 minutes. Dimethyl sulfide (13.8 mL, 187 mmol,6.1 equiv) was added and the solution was stirred at 20° C. for 20hours. After concentration 1-(triphenylphosphoranylidene)-2-propanone(19.5 g, 61.2 mmol, 2.0 equiv) and 150 mL of THF was added. The mixturewas continued to stir at 20° C. for 15 hours followed by concentration.The residue was purified by MPLC (hexanes:methyl tert-butylether=65%:35%) to give the product as a colorless oil (6.4 g, 54%). ¹HNMR (500 MHz, CDCl₃) δ 6.69 (dt, J=15.9 Hz, J=7.3 Hz, 1H), 6.13 (d,J=15.9 Hz, 1H), 5.15 (d, J=7.6 Hz, 1H), 4.91 (d, J=12.0 Hz, 1H), 4.66(d, J=12.0 Hz, 1H), 4.63-4.59 (m, 1H), 2.87-2.82 (m, 1H), 2.69-2.62 (m,1H), 2.22 (s, 3H), 1.41 (s, 9H). HRMS (ESI⁺): Calcd. For C₁₄H₂₀C₁₃NO₅Na([M+Na]⁺), 410.0299. Found: 410.0317.

(R)-2,2,2-Trichloroethyl 2-(tert-butoxycarbonylamino)-6-oxoheptanoate(MZ728)

A round-bottom flask was charged with MZ725 (6.30 g, 17.5 mmol, 1equiv.), Pd/C (10 wt. %, 740 mg, 0.695 mmol, 0.040 equiv.), EtOAc (54mL), and EtOH (6 mL) followed by setting a H₂ balloon on the top of theflask. The mixture was stirred under the H₂ atmosphere at 20° C. for 15hours and then filtered through a short celite pad. The solution wasconcentrated and the crude residue was purified by MPLC (hexanes:ethylacetate=70%:30%) to provide the product as a colorless oil (5.20 g,82%). ¹H NMR (500 MHz, CDCl₃) δ 5.06 (d, J=8.3 Hz, 1H), 4.92 (d, J=11.9Hz, 1H), 4.65 (d, J=11.9 Hz, 1H), 4.44-4.39 (m, 1H), 2.55-2.43 (m, 2H),2.13 (s, 3H), 1.92-1.84 (m, 1H), 1.74-1.65 (m, 3H), 1.45 (s, 9H). HRMS(ESI⁺): Calcd. For C₁₄H₂₂C₁₃NO₅Na ([M+Na]⁺), 412.0456. Found: 412.0471.

(R)-2,2,2-Trichloroethyl7-bromo-2-(tert-butoxycarbonylamino)-6-oxoheptanoate (MZ740)

n-Butyllithium solution (2.5 M in THF, 9.28 mL, 23.2 mmol, 3 equiv.) wasslowly added to a solution of 1,1,1,3,3,3-hexamethyldisilazane (4.93 mL,23.2 mmol, 3 equiv.) in 250 mL of THF at −78° C. under N₂. The solutionwas stirred at 0° C. for 20 minutes, cooled to −78° C. andchlorotrimethylsilane (9.90 mL, 77.4 mmol, 10 equiv.) was slowly addedfollowed by MZ442 (3.99 g, 10.65 mmol, 1 equiv.) as a THF solution (20mL). After 20 minutes anhydrous triethylamine (6.0 mL, 43.2 mmol, 5.6equiv.) was slowly added. The mixture was continued to stir for 5minutes and the reaction was quenched with 50 mL of saturated aqueousNaHCO₃ solution and extracted with methyl tert-butyl ether (3×100 mL).The organic layer was washed with brine (5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was dissolved in 10 mL of THF and thesolution was added to a stirred suspension of N-bromosuccinimide (1.38g, 7.74 mmol, 1.0 equiv.) and NaHCO₃ (780 mg, 9.29 mmol, 1.2 equiv.) inTHF (100 mL) at −78° C. under N₂. After stirring for 1.5 hour, thereaction was quenched by adding 30 mL of saturated aqueous NaHCO₃solution and the mixture was extracted with methyl tert-butyl ether(3×50 mL). The organic layer was washed with brine (5 mL), dried overMgSO₄ and concentrated. The crude residue was purified by MPLC(hexanes:ethyl acetate=85%:15%) to provide the product as a yellow oil(2.13 g, 61%). ¹H NMR (500 MHz, CDCl₃) δ 5.05 (d, J=8.3 Hz, 1H), 4.92(d, J=12.0 Hz, 1H), 4.65 (d, J=12.0 Hz, 1H), 4.46-4.39 (m, 1H), 3.87 (s,2H), 2.76-2.70 (m, 2H), 1.95-1.89 (m, 1H), 1.80-1.70 (m, 3H), 1.45 (s,9H). HRMS (ESI): Calcd. For C₁₄H₂₁BrCl₃NO₅Na ([M+Na]⁺), 489.9561. Found:489.9575.

2,2,2-Trichloroethyl(R)-5-(2-((R,E)-4-bromo-2-((triisopropylsilyl)oxy)pent-3-en-1-yl)thiazol-4-yl)-2-((tert-butoxycarbonyl)amino)pentanoate(MZ743)

Following the same procedure as in the literature, (Romo, D., Rzasa, R.M., Shea, H. A., Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., andLiu, J. O. (1998) Total Synthesis and Immunosuppressive Activity of(−)-Pateamine A and Related Compounds: Implementation of aβ-Lactam-Based Macrocyclization, J. Am. Chem. Soc. 120, 12237-12254) thereaction between MZ740 (2.00 g, 4.26 mmol, 1.1 equiv.) and Fragment C(1.47 g, 3.87 mmol, 1 equiv.) formed the product as a colorless oil(2.24 g, 79%). ¹H NMR (500 MHz, CDCl₃) δ 6.76 (s, 1H), 5.87-5.85 (m,1H), 5.09 (d, J=8.3 Hz, 1H), 4.92 (d, J=12 Hz, 1H), 4.77 (dt, J=8.9, 6.3Hz, 1H), 4.63 (d, J=12 Hz, 1H), 4.47-4.42 (m, 1H), 3.22 (dd, J=14.2 Hz,6.3 Hz, 1H), 3.10 (dd, J=14.2 Hz, 6.4 Hz, 1H), 2.81-2.71 (m, 2H), 2.09(s, 3H), 1.98-1.92 (m, 1H), 1.86-1.76 (m, 2H), 1.75-1.71 (m, 1H), 1.44(s, 9H), 1.06-0.97 (m, 21H). HRMS (ESI⁺): Calcd. For C₂₉H₄₉BrCl₃N₂O₅SSi([M+H]⁺), 749.1380. Found: 749.1386.

2,2,2-Trichloroethyl(R)-5-(2-((R,E)-4-bromo-2-hydroxypent-3-en-1-yl)thiazol-4-yl)-2-(tert-butoxycarbonylamino)pentanoate(MZ744)

To a solution of MZ743 (2.70 g, 3.59 mmol, 1.0 equiv.) in 180 mL of THFat −20° C. under N₂ was added a pre-mixed solution of tetrabutylammoniumfluoride (1 M in THF, 10.8 mL, 10.8 mmol, 3 equiv.) and acetic acid (495μL, 8.63 mmol, 2.4 equiv.) under N₂. The mixture was kept in a −20° C.freezer for 15 hours, diluted with 200 mL of methyl tert-butyl ether,washed with saturated aqueous NaHCO₃ solution (5 mL), water (5 mL) andbrine (5 mL). The organic layer was dried over MgSO₄ and concentrated invacuo. The crude residue was purified by MPLC (hexanes:EtOAc=1:1) togive the product as a colorless oil (1.75 g, 82%). ¹H NMR (500 MHz,CDCl₃) δ 6.78 (s, 1H), 5.95-5.92 (m, 1H), 5.12-5.09 (m, 1H), 4.91 (d,J=12.0 Hz, 1H), 4.73-4.69 (m, 1H), 4.61 (d, J=12.0 Hz, 1H), 4.45-4.41(m, 1H), 4.23 (brs, 1H), 3.11 (d, J=5.9 Hz, 2H), 2.82-2.72 (m, 2H), 2.27(s, 3H), 1.95-1.90 (m, 1H), 1.84-1.79 (m, 2H), 1.77-1.70 (m, 1H), 1.43(s, 9H). HRMS (ESI⁺): Calcd. For C₂₀H₂₉BrCl₃N₂O₅S ([M+H]⁺), 593.0046.Found: 593.0051.

(S,E)-4-Bromo-1-(4-((R)-4-(tert-butoxycarbonylamino)-5-oxo-5-(2,2,2-trichloroethoxy)pentyl)-3λ⁴-thiazol-2-yl)pent-3-en-2-yl(S,E)-5-methyl-7-(triisopropylsilyloxy)oct-4-en-2-ynoate (MZ747)

Following the same procedure as in the literature, (Romo, D., Choi, N.S., Li, S., Buchler, I., Shi, Z., and Liu, J. O. (2004) Evidence forSeparate Binding and Scaffolding Domains in the Immunosuppressive andAntitumor Marine Natural Product, Pateamine A: Design, Synthesis, andActivity Studies Leading to a Potent Simplified Derivative, J. Am. Chem.Soc. 126, 10582-10588.) MZ744 (1.30 g, 2.19 mmol, 1 equiv.) reacted withFragment F (852 mg, 2.63 mmol, 1.2 equiv.) to afford the product as acolorless oil (1.43 g, 73%). ¹H NMR (500 MHz, CDCl₃) δ 6.80 (s, 1H),5.89-5.87 (m, 1H), 5.78 (dt, J=9.6 Hz, 6.7 Hz, 1H), 5.41-5.40 (m, 1H),5.13 (d, J=8.2 Hz, 1H), 4.92 (d, J=12.0 Hz, 1H), 4.63 (d, J=12.0 Hz,1H), 4.46-4.42 (m, 1H), 4.15-4.09 (m, 1H), 3.38 (dd, J=14.8 Hz, 7.0 Hz,1H), 3.27 (dd, J=14.8 Hz, 6.4 Hz, 1H), 2.83-2.72 (m, 2H), 2.41 (dd,J=13.2 Hz, 5.3 Hz, 1H), 2.29 (d, J=1.2 Hz, 3H), 2.25 (dd, J=13.2 Hz, 6.9Hz, 1H), 2.01 (d, J=1.0 Hz, 3H), 1.96-1.91 (m, 1H), 1.86-1.80 (m, 2H),1.76-1.69 (m, 1H), 1.44 (s, 9H), 1.13 (d, J=6.0 Hz, 3H), 1.05 (s, 21H).HRMS (ESI⁺): Calcd. For C₃₈H₅₉BrCl₃N₂O₇SSi ([M+H]⁺), 901.2041. Found:901.2040.

(S,E)-4-Bromo-1-(4-((R)-4-(tert-butoxycarbonylamino)-5-oxo-5-(2,2,2-trichloroethoxy)pentyl)thiazol-2-yl)pent-3-en-2-yl (S,E)-7-hydroxy-5-methyloct-4-en-2-ynoate(MZ533)

Tetrabutylammonium fluoride (1 M in THF, 5 mL, 5.0 mmol) was mixed withHF/py (70 w/w % HF, 138 μL, 5.3 mmol) at 0° C. and 2.16 mL of themixture was added to a solution of MZ747 (290 mg, 0.322 mmol) in THF(8.3 mL) at 0° C. The reaction was maintained at 4° C. for 15 hours. Themixture was diluted with 100 mL of EtOAc and was washed with saturatedaqueous NaHCO₃ solution (5 mL), water (5 mL), and brine (5 mL). Theorganic layer was dried over MgSO₄ and concentrated. The crude residuewas purified by MPLC (heaxnes:EtOAc=4:1) to give the desired product asa colorless oil (240 mg, 76%). ¹H NMR (500 MHz, CDCl₃) δ 6.79 (s, 1H),5.88-5.85 (m, 1H), 5.77 (dt, J=9.7 Hz, 6.3 Hz, 1H), 5.45-5.44 (m, 1H),5.18 (d, J=8.3 Hz, 1H), 4.90 (d, J=12.0 Hz, 1H), 4.61 (d, J=12.0 Hz,1H), 4.44-4.40 (m, 1H), 4.02-3.96 (m, 1H), 3.36 (dd, J=14.8 Hz, 7.0 Hz,1H), 3.27 (dd, J=14.8 Hz, 6.1 Hz, 1H), 2.82-2.71 (m, 2H), 2.32-2.23 (m,2H), 2.28 (d, J=1.0 Hz, 3H), 2.01 (d, J=0.9 Hz, 3H), 1.96-1.88 (m, 1H),1.84-1.78 (m, 2H), 1.74-1.67 (m, 1H), 1.42 (s, 9H), 1.20 (d, J=6.2 Hz,3H). OH not observed. HRMS (ESI⁺): Calcd. For C₂₉H₃₉BrCl₃N₂O₇S ([M+H]⁺),743.0727. Found: 743.0728.

(R)-5-(2-((S,E)-4-Bromo-2-(((S,E)-7-hydroxy-5-methyloct-4-en-2-ynoyl)oxy)pent-3-en-1-yl)thiazol-4-yl)-2-(tert-butoxycarbonylamino)pentanoic acid (MZ749)

Following the known procedure, (Romo, D., Rzasa, R. M., Shea, H. A.,Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., and Liu, J. O. (1998)Total Synthesis and Immunosuppressive Activity of (−)-Pateamine A andRelated Compounds: Implementation of a β-Lactam-Based Macrocyclization,J. Am. Chem. Soc. 120, 12237-12254) MZ749 was synthesized from MZ748(180 mg, 0.242 mmol) as a colorless oil (123 mg, 83%). ¹H NMR (500 MHz,CDCl₃) δ 6.84 (s, 1H), 5.87-5.84 (m, 1H), 5.79-5.74 (m, 1H), 5.44 (s,1H), 5.31 (d, J=7.8 Hz, 1H), 4.33-4.29 (m, 1H), 4.03-3.97 (m, 1H), 3.39(dd, J=14.8 Hz, 7.6 Hz, 1H), 3.34 (dd, J=14.8 Hz, 5.9 Hz, 1H), 2.80-2.72(m, 2H), 2.31-2.24 (m, 2H), 2.27 (d, J=1.2 Hz, 3H), 1.99 (s, 3H),1.87-1.83 (m, 1H), 1.78-1.69 (m, 3H), 1.42 (s, 9H), 1.21 (d, J=6.2 Hz,3H). OH not observed. HRMS (ESI⁺): Calcd. For C₂₇H₃₈BrN₂O₇S ([M+H]⁺),613.1583. Found: 613.1588.

tert-Butyl((1²Z,3S,8E,11S,14R)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1 (2,4)-thiazolacycloheptadecaphane-6-yn-8-en-14-yl)carbamate(MZ750)

Following the known procedure (Romo, D., Choi, N. S., Li, S., Buchler,I., Shi, Z., and Liu, J. O. (2004) Evidence for Separate Binding andScaffolding Domains in the Immunosuppressive and Antitumor MarineNatural Product, Pateamine A: Design, Synthesis, and Activity StudiesLeading to a Potent Simplified Derivative, J. Am. Chem. Soc. 126,10582-10588.) but the reaction time was extended to 45 hours, MZ749(38.0 mg, 0.0619 mmol) afforded the desired product as a colorless oil(17.5 mg, 46%). ¹H NMR (500 MHz, CDCl₃) δ 6.82 (s, 1H), 6.03-6.00 (m,1H), 5.96-5.91 (m, 1H), 5.33 (s, 1H), 5.26-5.20 (m, 1H), 4.70 (d, J=8.3Hz, 1H), 4.30-4.26 (m, 1H), 3.33-3.26 (m, 2H), 2.80-2.75 (m, 1H),2.71-2.66 (m, 1H), 2.43 (d, J=1.3 Hz, 3H), 2.34 (d, J=7.2 Hz, 2H),1.93-1.87 (m, 1H), 1.89 (s, 3H), 1.72-1.61 (m, 3H), 1.45 (s, 9H), 1.27(d, J=6.0 Hz, 3H). HRMS (ESI⁺): Calcd. For C₂₇H₃₆BrN₂O₆S ([M+H]⁺),595.1477. Found: 595.1483.

tert-Butyl((1²Z,3S,6Z,8E,11S,14R)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1 (2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)carbamate(MZ751)

A H₂ balloon was placed on the top of a flask containing MZ750 (18 mg,0.030 mmol, 1 equiv.), lindlar catalyst (9.0 mg), and MeOH (2 mL). Themixture was stirred at 20° C. for 3 hours until no starting materialvisible on TLC and was filtered through a cotton pad which was rinsedwith 5 mL of EtOAc. The solvents were evaporated in vacuo and the cruderesidue was purified on a silica gel chromatography (hexanes:EtOAc=5:1)to provide the product as a colorless oil (11 mg, 61%). ¹H NMR (500 MHz,CDCl₃) δ 6.93 (d, J=11.7 Hz, 1H), 6.71 (t, J=11.7 Hz, 1H), 6.67 (s, 1H),6.02 (t, J=11.5 Hz, 1H), 5.96 (d, J=9.6 Hz, 1H), 5.72 (d, J=9.7 Hz, 1H),5.35 (d, J=11.3 Hz, 1H), 5.01-4.95 (m, 1H), 4.16 (t, J=11.1 Hz, 1H),3.26 (dd, J=14.3 Hz, 2.1 Hz, 1H), 3.14 (dd, J=14.3 Hz, 11.3 Hz, 1H),2.97-2.93 (m, 1H), 2.59-2.53 (m, 1H), 2.48 (s, 3H), 2.33 (dd, J=12.7 Hz,J=11.1 Hz, 1H), 2.25-2.20 (m, 1H), 2.10 (d, J=12.7 Hz, 1H), 1.81 (s,3H), 1.73-1.64 (m, 3H), 1.45 (s, 9H), 1.24 (d, J=6.3 Hz, 3H). HRMS(ESI⁺): Calcd. For C₂₇H₃₈BrN₂O₆S ([M+H]⁺), 597.1634. Found: 597.1637.

(1²Z,3S,6Z,8E,11S,14R)-14-Amino-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-diene-5,13-dione(MZ752)

A solution of trifluoroacetic acid (0.3 mL) in DCM (1.5 mL) was cooledto 0° C. and added to MZ554 (12.0 mg, 0.020 mmol) at 0° C. under N₂. Thereaction was kept in a 4° C. refrigerator for 18 hours. Toluene (10 mL)was added and the solvents were evaporated in vacuo with the water bathwas kept at 20° C. The crude residue was purified on a silica gelchromatography (DCM:MeOH=20:1) to give the product as a colorless oil inthe form of a TFA salt (12 mg, 98%). ¹H NMR (500 MHz, CDCl₃) δ 6.81 (s,1H), 6.79 (t, J=11.5 Hz, 1H), 6.72 (s, 1H), 6.2 (td, J=10.1 Hz, 2.8 Hz,1H), 5.95 (d, J=9.4 Hz, 1H), 5.46 (d, J=9.1 Hz, 1H), 5.14-5.08 (m, 1H),4.12-4.09 (m, 1H), 3.24 (dd, J=14.9 Hz, 2.8 Hz, 1H), 3.17 (dd, J=14.9Hz, 10.6 Hz, 1H), 2.91-2.87 (m, 1H), 2.62-2.57 (m, 1H), 2.53 (dd, J=13.1Hz, 11.2 Hz, 1H), 2.43 (s, 3H), 2.23-2.15 (m, 1H), 2.19 (d, J=13.1 Hz,1H), 1.89-1.80 (m, 2H), 1.83 (s, 3H), 1.77-1.70 (m, 1H), 1.33 (d, J=6.2Hz, 3H). NH₃ ⁺ not observed. HRMS (ESI⁺): Calcd. For C₂₂H₃₀BrN₂O₄S([M+H]⁺), 497.1110. Found: 497.1122.

1,1,1-Trichloro-2-methylpropan-2-yl((1²Z,3S,6Z,8E,11S,14R)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-1⁴-yl)carbamate (MZ754)

Following the known procedure, (Romo, D., Rzasa, R. M., Shea, H. A.,Park, K., Langenhan, J. M., Sun, L., Akhiezer, A., and Liu, J. O. (1998)Total Synthesis and Immunosuppressive Activity of (−)-Pateamine A andRelated Compounds: Implementation of a β-Lactam-Based Macrocyclization,J. Am. Chem. Soc. 120, 12237-12254) MZ752 (6.7 mg, 0.0014 mmol) affordedthe desired product as a colorless oil (8.0 mg, 85%). ¹H NMR indicates amixture of rotamers (3:1). The major rotamer: ¹H NMR (600 MHz, CDCl₃) δ6.83 (d, J=11.7 Hz, 1H), 6.69 (t, J=11.7 Hz, 1H), 6.68 (s, 1H),6.15-6.11 (m, 1H), 6.03 (d, J=9.8 Hz, 1H), 5.95 (d, J=9.0 Hz, 1H), 5.37(d, J=11.7 Hz, 1H), 5.00-4.95 (m, 1H), 4.19-4.15 (m, 1H), 3.26 (dd,J=14.3 Hz, 2.7 Hz, 1H), 3.12 (J=14.3 Hz, 11.4 Hz, 1H), 3.01-2.98 (m,1H), 2.59-2.55 (m, 1H), 2.46 (s, 3H), 2.30 (dd, J=13.1 Hz, 11.2 Hz, 1H),2.27-2.22 (m, 1H), 2.11 (d, J=13.1 Hz, 1H), 1.95 (s, 6H, 2CH₃), 1.82 (s,3H), 1.75-1.64 (m, 3H), 1.25 (d, J=6.2 Hz, 3H). HRMS (ESI⁺): Calcd. ForC₂₇H₃₅BrCl₃N₂O₆S ([M+H]⁺), 699.0465. Found: 699.0457.

1,1,1-Trichloro-2-methylpropan-2-yl((1²Z,3S,6Z,8E,11S,14R)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazol acycloheptadecaphane-6,8-dien-14-yl)carbamate (MZ756)

The coupling between MZ754 (2.3 mg, 0.0033 mmol, 1 equiv.) and FragmentG (2.7 mg, 0.0066 mmol, 2 equiv.) followed the known procedure. Low, W.K., Li, J., Zhu, M., Kommaraju, S. S., Shah-Mittal, J., Hull, K., Liu,J. O., and Romo, D. (2014) Second-generation derivatives of theeukaryotic translation initiation inhibitor pateamine A targeting eIF4 Åas potential anticancer agents, Bioorg. Med. Chem. 22, 116-125. Thereaction mixture was kept at 20° C. for 15 hours and transferreddirectly to a silica gel chromatography for purification(dichloromethane:MeOH:triethylamine=50:1:0.1). The product was furtherpurified by the Prep-HPLC (solvent A: H₂O buffered with 8 mM HCOOH and12 mM NH₃,H₂O, pH=9.0; solvent B: CH₃CN/H₂O (9:1 v/v) buffered with 8 mMHCOOH and 12 mM NH₃,H₂O; isocratic elution, solvent A/solvent B=1:4).The collected fractions were concentrated to give a mixture of theproduct and solid ammonium formate, upon which DCM (10 mL) was added andthe suspension was filtered through a sintered Buchner glass funnel. Theprecipitates were rinsed with extra dichloromethane (2×5 mL). Afterconcentration in vacuo the product was obtained as a colorless oil inthe form a salt with formic acid (2.0 mg, 48%). ¹H NMR indicates amixture of rotamers (4:1). Major rotamer: ¹H NMR (600 MHz, CDCl₃) δ 8.56(brs, 1H), 6.83 (d, J=11.6 Hz, 1H), 6.67 (s, 1H), 6.66 (t, J=11.6 Hz,1H), 6.41 (d, J=15.9 Hz, 1H), 6.35 (ddd, J=11.5 Hz, 8.8 Hz, 2.9 Hz, 1H),6.26 (d, J=16.0 Hz, 1H), 6.16 (d, J=9.7 Hz, 1H), 5.69 (t, J=7.1 Hz, 1H),5.55 (d, J=8.5 Hz, 1H), 5.39 (d, J=11.6 Hz, 1H), 4.99-4.95 (m, 1H),4.19-4.15 (m, 1H), 3.28-3.24 (m, 2H), 3.12 (dd, J=14.1 Hz, 11.4 Hz, 1H),3.02-2.99 (m, 1H), 2.59-2.54 (m, 1H), 2.39 (s, 6H), 2.32-2.25 (m, 1H),2.27-2.22 (m, 1H), 2.09 (d, J=12.9 Hz, 1H), 2.01-1.95 (m, 1H), 1.99 (s,3H), 1.95 (s, 6H, 2CH₃), 1.84 (s, 3H), 1.80 (s, 3H), 1.76-1.65 (m, 3H),1.25 (d, J=6.2 Hz, 3H). HRMS (ESI⁺): Calcd. For C₃₅H₄₉C₁₃N₃O₆S ([M+H]⁺),744.2408. Found: 744.2338.

N-((1²Z,3S,6Z,8E,11S,14R)-3-((E)-2-bromoprop-1-en-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl) acetamide (MZ753). Toa solution of MZ752 (10.5 mg, 0.015 mmol, 1 equiv.) in 0.5 mL of CH₂Cl₂were added pyridine (0.25 mL, excess) and AcCl (6.2 μL, 0.09 mmol, 6equiv.) at 0° C. The reaction was continued at 4° C. for 15 hours. Themixture was diluted with 25 mL of EtOAc, washed with saturated aq.NaHCO₃, H₂O and brine. The organic phase was dried over MgSO₄ andconcentrated in vacuo. The residue was purified by a flashchromatography (hexanes:acetone=2:1) to give the desired product as acolorless oil (7.5 mg, 81%). ¹H NMR (500 MHz, CDCl₃) δ 6.90 (d, J=11.6Hz, 1H), 6.88 (d, J=8.9 Hz, 1H), 6.76 (t, J=11.6 Hz, 1H), 6.68 (s, 1H),6.12 (ddd, J=11.1 Hz, 9.7 Hz, 2.8 Hz, 1H), 5.89-5.96 (m, 1H), 5.38 (d,J=11.6 Hz, 1H), 5.00-4.95 (m, 1H), 4.59 (ddd, J=11.9 Hz, 9.4 Hz, 2.3 Hz,1H), 3.25 (dd, J=14.4 Hz, 2.9 Hz, 1H), 3.16 (dd, J=14.4 Hz, 11.0 Hz,1H), 2.96-2.92 (m, 1H), 2.57-2.51 (m, 1H), 2.46 (d, J=1.3 Hz, 3H), 2.33(dd, J=13.2 Hz, 10.9 Hz, 1H), 2.12 (d, J=13.2 Hz, 1H), 2.11 (s, 3H),1.93-1.87 (m, 1H), 1.85-1.80 (m, 1H), 1.83 (s, 3H), 1.73-1.65 (m, 2H),1.25 (d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. For C₂₄H₃₂BrN₂O₅S ([M+H]⁺),539.1215. Found: 539.1229.

N-((1²Z,3S,6Z,8E,11S,14R)-3-((1E,3E,5E)-7-(dimethylamino)-2,5-dimethylhepta-1,3,5-trien-1-yl)-9,11-dimethyl-5,13-dioxo-4,12-dioxa-1(2,4)-thiazolacycloheptadecaphane-6,8-dien-14-yl)acetamide (MZ757). The coupling between MZ753 (3.6 mg, 0.0067 mmol, 1equiv.) and Fragment G (5.5 mg, 0.013 mmol, 2 equiv.) was based on theknown procedure. Low, W. K., Li, J., Zhu, M., Kommaraju, S. S.,Shah-Mittal, J., Hull, K., Liu, J. O., and Romo, D. (2014)Second-generation derivatives of the eukaryotic translation initiationinhibitor pateamine A targeting eIF4 Å as potential anticancer agents,Bioorg. Med. Chem. 22, 116-125. The purification procedure was the sameas that of MZ756, but using a different prep-HPLC condition (solvent A:H₂O buffered with 8 mM HCOOH and 12 mM NH₃,H₂O, pH=9.0; solvent B:methanol; gradient elution, solvent A/solvent B=2:3-1:4 within 12minutes) gave the pure product in the form of salt with formic acid as acolorless oil (2.5 mg, 53%). ¹H NMR (600 MHz, CDCl₃) δ 8.44 (brs, 1H),6.99 (d, J=9.5 Hz, 1H), 6.91 (d, J=11.6 Hz, 1H), 6.74 (t, J=11.6 Hz,1H), 6.68 (s, 1H), 6.40 (d, J=15.7 Hz, 1H), 6.35 (ddd, J=11.5 Hz, 9.4Hz, 3.0 Hz, 1H), 6.33 (d, J=15.7 Hz, 1H), 5.69 (t, J=7.1 Hz, 1H), 5.60(d, J=9.1 Hz, 1H), 5.40 (d, J=11.6 Hz, 1H), 5.00-4.95 (m, 1H), 4.61(ddd, J=11.9 Hz, 9.7 Hz, 2.3 Hz, 1H), 3.62 (d, J=7.6 Hz, 2H), 3.25 (dd,J=14.5 Hz, 2.9 Hz, 1H), 3.17 (dd, J=14.5 Hz, J=11.2 Hz, 1H), 2.98-2.94(m, 1H), 2.62 (s, 6H), 2.57-2.52 (m, 1H), 2.43 (dd, J=13.2 Hz, 10.8 Hz,1H), 2.16 (s, 3H), 2.14-2.08 (m, 1H), 2.11 (d, J=13.2 Hz, 1H), 2.02 (s,3H), 1.87 (s, 3H), 1.85-1.80 (m, 1H), 1.82 (s, 3H), 1.72-1.66 (m, 2H),1.25 (d, J=6.3 Hz, 3H). HRMS (ESI⁺): Calcd. For C₃₂H₄₆N₃O₅S ([M+H]⁺),584.3158. Found: 584.3107.

Example 6 Plasma Protein Binding Assay

In this example, a representative plasma protein binding (PPB) assayused to provide the plasma protein binding data is described.

The assay includes the following steps.

1. Frozen plasma was thawed and centrifuged at 1120 g for 10 min toremove any particulates. The plasma was decanted, the pH was measuredand, if required, was adjusted to pH7.4 with lactic acid.

2. All compounds were prepared at 10 mM concentration in DMSO. Thesolutions were carefully vortexed to ensure the compounds dissolved. The10 mM DMSO solutions were diluted to 500 μM MeOH solutions (5+95 μl).

3. In a 2 mL 96-well plate (DWP), 1000 μL of plasma was pipetted intoeach well in Columns 1-4.

4. 10 μL of the 500 μM compound solution was pipetted to 1000 μL ofcorresponding plasma. (The final DMSO=0.05%). The solution was cappedand carefully vortexed for 5 min.

5. 200 μL of PBS buffer was added to each receiver well of the dialysisplate (buffer was added to the Receiver first).

6. The bottom of dialysis plate was capped and turned over to the orangedonor side (top) of dialysis plate.

7. The orange donor side of the dialysis plate was uncapped and 200 μLof the 10 μM drug/plasma samples were transferred from the 2 ml 96-DWPto the corresponding donor wells in the dialysis plate. The orange donorside of the dialysis plate was then capped.

8. The dialysis plate was placed onto the plate rotator in 37° C. ovenand incubated at 37° C. with a rotation speed 20 rpm for 22 hr.

9. To a 1 mL 96-DWP, 50 μL/well of the 5 μM drug/plasma samples waspipetted from the 2 mL 96-DWP in triplicate. 50 μL/well of PBS and then300 μL of ACN/IS was added. The plate was kept at 4° C. This plateserves as the recovery plate.

10. Two 1 mL 96-DWP were prepared and marked as Donor Plate and ReceiverPlate.

11. The dialysis plate was removed from the 37° C. oven.

12. The caps were removed from the donor side of the dialysis plate.

13. 50 μL of samples were pipetted from the donor side of the dialysisplate and added into the 96-DWP Donor Plate. 50 μL of PBS buffer wasadded into the 96-DWP Donor Plate.

14. The donor side of the dialysis plate was capped and turned over.Caps were removed from the Receiver side wells. 0 uL of samples werepipetted from the Receiver side of the dialysis plate and added to the96-DWP Receiver Plate.

15. 50 μL of blank plasma was added into the 96-DWP Receiver Plate.

16. 300 μL/well of 1 μM of imipramine (IS) in acetonitrile (ACN) wasadded into the 96-DWP Donor Plate, the 96-DWP Receiver Plate, and theRecovery Plate.

17. The three plates were capped and vortexed for 10 minutes.

18. The plates were centrifuged at 4° C. at 4000 rpm for 10 min.

19. 150 μL of quench samples were transferred from each plate to acorresponding 96-DWP injection plate.

20. 150 μL of 0.1% acetic acid/water was added to the injection plates.

21. The injection plates were capped and vortexed for 5 min.

22. The plates were centrifuged at 4° C. at 4000 rpm for 5 min.

23. The samples in the Receiver Plate, Donor Plate, and Recovery Platewere analyzed by LC/MS/MS

HPLC: Agilent 1290 infinity binary LC/HTC injector. Column:Sigma-Aldrich Supelco Ascentis fused-core C18, 2.7 um, 2.1×20 mm.Solvent A: 0.1% acetic acid/water. Solvent B: 0.1% aceticacid/acetonitrile. Column temperature: 40° C. Injection volume: 2 uL.Time/flow rate: 0 min/0.5 mL/min 11.3 min/0.5 mL/min 11.31 min/1.0mL/min 11.7 min/1.0 mL/min.

MS/MS: Agilent 6460, Positive, ESI. Sheath gas temperature: 400° C.Sheath gas flow: 12 L/min. Gas temperature: 300° C. Gas flow: 11 L/min.Capillary voltage: 4000 V. Nozzle voltage: 500 V. Nebulizer: 35 psi.Cell accelerator voltage: 7 V.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

The invention claimed is:
 1. A compound having the formula:

or a stereoisomer, racemate, or a pharmaceutically acceptable saltthereof, wherein Z is selected from R and OR′, wherein R is selectedfrom C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl, and C3-C12 alkyl groupsin which one or more carbons are replaced with O, NH, or N(CH₃), andwherein R¹ is selected from hydrogen, C1-C6 alkyl, C1-C6 haloalkyl,C6-C10 aryl, and C3-C12 alkyl groups in which one or more carbons arereplaced with O, NH, or N(CH₃).
 2. A compound of claim 1 having theformula:

or a pharmaceutically acceptable salt thereof, wherein Z is selectedfrom R and OR¹, wherein R is selected from C1-C6 alkyl, C1-C6 haloalkyl,C6-C10 aryl, and C3-C12 alkyl groups in which one or more carbons arereplaced with O, NH, or N(CH₃), and wherein R¹ is selected fromhydrogen, C1-C6 alkyl, C1-C6 haloalkyl, C6-C10 aryl, and C3-C12 alkylgroups in which one or more carbons are replaced with O, NH, or N(CH₃).3. The compound of claim 1 having the formula:

wherein A⁻ is a pharmaceutically acceptable counter ion.
 4. The compoundof claim 2 having the formula:

wherein A⁻ is a pharmaceutically acceptable counter ion.
 5. The compoundof claim 3, wherein A⁻ is selected from the group consisting ofchloride, bromide, iodide, sulfate, phosphate, formate, acetate,trifluoroacetate, maleate, fumarate, succinate, tartrate, oxalate,citrate, malate, benzoate, toluenesulfonate, methanesulfonate, andbenzenesulfonate.
 6. The compound of claim 1, wherein Z is C1-C6 alkyl.7. The compound of claim 1, wherein Z is methyl.
 8. The compound ofclaim 1, wherein Z is C1-C6 haloalkyl.
 9. The compound of claim 1,wherein Z is trifluoromethyl.
 10. The compound of claim 1, wherein Z isC6-C10 aryl.
 11. The compound of claim 1, wherein Z is phenyl.
 12. Thecompound of claim 1, wherein Z is C3-C12 alkyl in which one or morecarbons are replaced with O, NH, or N(CH₃).
 13. The compound of claim 1,wherein Z is selected from CH₂—O—CH₃, CH₂CH₂—O—CH₃, and—CH₂CH₂—O—CH₂CH₂—O—CH₃.
 14. The compound of claim 1, wherein Z isselected from —CH₂—NH—CH₃, —CH₂—N(CH₃)₂, —CH₂CH₂—NH—CH₃, and—CH₂CH₂—N(CH₃)₂.
 15. A pharmaceutical composition, comprising a compoundof claim 1, or a stereoisomer or a pharmaceutically acceptable saltthereof, and a pharmaceutically acceptable carrier.
 16. An antibodyconjugate for the delivery of an α-amino pateamine derivative,comprising a compound of claim 1, or a stereoisomer or apharmaceutically acceptable salt thereof, covalently coupled directly orthrough a linker unit to an antibody or functional fragment thereof,wherein the functional fragment is selected from the group consisting ofFab, Fab′, F(ab′)2, and Fv fragments; linear antibodies; single-chainantibody molecules; an scFv; an IgG ΔCH2, a minibody, a diabody, atriabody, a tetrabody, a dsFv; an sc-Fv-Fc; an (scFv)2; a fragmentproduced by a Fab expression library; an anti-idiotypic (anti-Id)antibody; and multispecific antibodies formed from antibody fragment(s).17. A pharmaceutical composition, comprising the antibody conjugate ofclaim 16 and a pharmaceutically acceptable carrier.
 18. A method forinhibiting growth of chronic lymphocytic leukemia (CLL) cells,comprising contacting CLL cells with a compound of claim 1, or astereoisomer or a pharmaceutically acceptable salt thereof.
 19. A methodfor inhibiting growth of chronic lymphocytic leukemia (CLL) cells,comprising contacting CLL cells with an antibody conjugate of claim 16.20. A method for treating chronic lymphocytic leukemia (CLL), comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a compound of claim 1, or a stereoisomer or a pharmaceuticallyacceptable salt thereof.
 21. A method for treating chronic lymphocyticleukemia (CLL), comprising administering to a subject in need thereof atherapeutically effective amount of a compound an antibody conjugate ofclaim 16.